Masahiro Itoh

Combined treatment with bortezomib plus bafilomycin A1 enhances the cytocidal effect and induces endoplasmic reticulum stress in U266 myeloma cells: crosstalk among proteasome, autophagy-lysosome and ER stress.

Bortezomib (BZ), a first line 26S proteasome inhibitor, induces a potent cytocidal effect with caspase-3 activation in multiple myeloma (MM) cell lines. Since IκBα is a substrate of the proteasome, the initial rationale for using BZ in MM has been to inhibit NF-κB. However, BZ rather activated NF-κB activity in U266 cells. BZ induces autophagy as well as endoplasmic reticulum (ER) stress in various cell lines tested. Inhibition of initial autophagosome formation by treatment with either 3-methyladenine or siRNA for LC3B in U266 cells and knockdown of the atg5 gene in a murine embryonic fibroblastic cell line all resulted in attenuation of BZ-induced cell death. In contrast, combined treatment with BZ and bafilomycin A1 (BAF), which is a specific inhibitor of vacuolar-ATPase and is used as an autophagy inhibitor at the late stage, resulted in synergistic cytotoxicity, compared with that by either BZ or BAF alone. BAF treatment also induced ER stress, but the kinetics of inductions of ER stress-related genes [e.g. CHOP (GADD153) and GRP78] completely differed between BZ- and BAF-treatments: BZ induced these ER stress markers within 8 h, whereas treatment with BAF required more than 48 h in U266 cells. In order to synchronize ER stress, we pre-treated U266 cells with BAF for 48 h, followed with BZ for 48 h. The sequential treatment with BAF and BZ induced a further enhanced cytotoxicity, compared with the simultaneous combination of BAF and BZ. These data suggest crosstalk among the ubiquitin-proteasome system, the autophagy-lysosome system, and ER stress. Controlling these interactions and kinetics appears to have important implications for optimizing clinical cancer treatment including MM-therapy.

Stable Chromosome Aberrations in Atomic Bomb Survivors: Results from 25 Years of Investigation

Abstract Kodama, Y., Pawel, D., Nakamura, N., Preston, D., Honda, T., Itoh, M., Nakano, M., Ohtaki, K., Funamoto, S. and Awa, A. A. Stable Chromosome Aberrations in Atomic Bomb Survivors: Results from 25 Years of Investigation. Radiat. Res. 156, 337–346 (2001). Frequencies of stable chromosome aberrations from more than 3,000 atomic bomb survivors were used to examine the nature of the radiation dose response. The end point was the proportion of cells with at least one translocation or inversion detected in Giemsa-stained cultures of approximately 100 lymphocytes per person. The statistical methods allow for both imprecision of individual dose estimates and extra-binomial variation. A highly significant and nonlinear dose response was seen. The shape of the dose response was concave upward for doses below 1.5 Sv but exhibited some leveling off at higher doses. This curvature was similar for the two cities, with a crossover dose (i.e. the ratio of the linear coefficient to the quadratic coefficient) of 1.7 Sv (95% CI 0.9, 4). The low-dose slopes for the two cities differed significantly: 6.6% per Sv (95% CI 5.5, 8.4) in Hiroshima and 3.7% (95% CI 2.6, 4.9) in Nagasaki. This difference was reduced considerably, but not eliminated, when the comparison was limited to people who were exposed in houses or tenements. Nagasaki survivors exposed in factories, as well as people in either city who were outside with little or no shielding, had a lower dose response than those exposed in houses. This suggests that doses for Nagasaki factory worker survivors may be overestimated by the DS86, apparently by about 60%. Even though factory workers constitute about 20% of Nagasaki survivors with dose estimates in the range of 0.5 to 2 Sv, calculations indicate that the dosimetry problems for these people have little impact on cancer risk estimates for Nagasaki.

Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.

A new murine model of autoimmune orchitis induced by immunization with viable syngeneic testicular germ cells alone. I, Immunological and histological studies

Experimental autoimmune orchitis (EAO) was produced in C3H/He mice with as high as 100% incidence by two or three s.c. injections of 1 × 107 viable syngeneic testicular germ cells (TC) without resorting to adjuvants, Bordetella pertussis vaccine, or other immunological manipulations. On day 40 after the first injection of TC. the lesions induced were characterized by interstitial infiltration of inflammatory cells and severe hypospermatogenesis in the testis with resulting whole organ atrophy and. in contrast, by a complete lack of epididymitis. Immunological studies revealed that this form of immunization caused both delayed‐type hypersensitivity and humoral antibody responses to syngeneic TC. We compared the susceptibilities to the induction of this type of EAO among six different strains of inbred mice comprising A/J, AKR, BALB/C, C3H/He, C57BL/6 and DBA/2 mice. All strains except for DBA/2 mice developed lesions of EAO to a greater or lesser extent, and severe disease was induced with high frequency in two strains, C3H/He and A/J. As this murine model of EAO can be induced without the use of Freunds complete adjuvant and B. pertussis vaccine, it is simply ‘autoimmune’ in nature and may provide new ways for further investigation into the immunological mechanisms which regulate deleterious autoimmune reactions to germ cell antigens leading to the male infertility.

Human Fetuses do not Register Chromosome Damage Inflicted by Radiation Exposure in Lymphoid Precursor Cells except for a Small but Significant Effect at Low Doses

Abstract Ohtaki, K., Kodama, Y., Nakano, M., Itoh, M., Awa, A. A., Cologne, J. and Nakamura, N. Human Fetuses do not Register Chromosome Damage Inflicted by Radiation Exposure in Lymphoid Precursor Cells except for a Small but Significant Effect at Low Doses. Radiat. Res. 161, 373–379 (2004). Human fetuses are thought to be highly sensitive to radiation exposure because diagnostic low-dose X rays have been suggested to increase the risk of childhood leukemia. However, animal studies generally have not demonstrated a high radiosensitivity of fetuses, and the underlying causes for the discrepancy remain unidentified. We examined atomic bomb survivors exposed in utero for translocation frequencies in blood lymphocytes at 40 years of age. Contrary to our expectation of a greater radiosensitivity in fetuses than in adults, the frequency did not increase with dose except for a small increase (less than 1%) at doses below 0.1 Sv, which was statistically significant. We interpret the results as indicating that fetal lymphoid precursor cells comprise two subpopulations. One is small in number, sensitive to the induction of both translocations and cell killing, but rapidly diminishing above 50 mSv. The other is the major fraction but is insensitive to registering damage expressed as chromosome aberrations. Our results provide a biological basis for resolving the long-standing controversy that a substantial risk of childhood leukemia is implicated in human fetuses exposed to low-dose X rays whereas animal studies involving mainly high-dose exposures generally do not confirm it.

Essential pathogenic role for endogenous interferon‐gamma (IFN‐γ) during disease onset phase of murine experimental autoimmune orchitis. I. In vivo studies

We previously found that immunization of CH3/He male mice with syngeneic testicular germ cells (TGC) without the aid of any adjuvants was sufficient to induce DTH to TGC and experimental autoimmune orchitis (EAO). To evaluate the role of endogenous IFN‐γ in this model, C3H/He mice immunized subcutaneously with TGC on days 0 and 14 received a single injection of anti‐murine IFN‐γ MoAb on day 15, 20 or 25. On day 45, DTH to TGC was tested, testis specimens were collected for histological examination, and blood samples collected for IFN‐γ measurement. The results showed that whilst MoAb treatment on day 15 or 25 did not influence DTH responses, EAO development, and appearance of IFN‐γ in the circulation, treatment on day 20 significantly suppressed all of them. Thus, a single injection with anti‐IFN‐γ MoAb may successfully down‐regulate testicular autoimmunity, provided that the treatment is given at an optimal time point during disease development.

A quantitative study of the effects of prenatal X-irradiation on the development of cerebral cortex in rats

Pregnant rats were exposed to a single whole body X-irradiation on day 15 of gestation at a dose of 1.0 Gy. The offspring showed microcephaly at 7 weeks of age. Their body weight, brain weight, cortical thickness and the numerical density of neurons and synapses in the somatosensory and visual cortex were examined. Significant decreases in cortical thickness in both somatosensory (25%) and visual (16%) cortex were observed. However, there were no significant changes in the numerical density of neurons and synapses, nor in synapse-to-neuron ratios in both cortical regions between control and X-irradiated groups. These results suggest that prenatal X-irradiation can decrease the number of neurons, and the neurons which survive X-irradiation proliferate and elaborate connections in a normal fashion. This is in contrast to the animals exposed to ochratoxin A, in which numerical density of neurons in the somatosensory cortex is increased, with normal numerical density of synapses, resulting in low synapse-to-neuron ratios. The discrepancy in the synapse-to-neuron ratios between the X-irradiation and ochratoxin A-treatment might derive from a different effect on the developing neurons.

A New Murine Model of Autoimmune Orchitis Induced by Immunization With Viable Syngeneic Testicular Germ Cells Alone.: II. Immunohistochemical Findings of Fully-Developed Inflammatory Lesion

Previous studies demonstrated that experimental autoimmune orchitis (EAO) was produced in C3H/He mice with very high incidence by two or three subcutaneous injections of viable syngeneic testicular germ cells, without the use of any adjuvants or immunopotentiators and that the disease induced was characterized by a complete lack of epididymitis despite a definite orchitis with hypospermatogenesis. In this report, immunohistochemical characterization of immune cells in the fully-developed orchitic lesion was carried out using monoclonal antibodies and immunoperoxidase staining. Thy-1.2+ cells, Mac-1+ cells, B220+ cells and cytoplasmic Ig-bearing cells in the lesion were estimated to be approximately 30, 15, 20 and 30% of all inflammatory cells, respectively. Major phenotype of T cells in the lesion was CD4+ (approximately 85%) with the remainder (approximately 15%) being CD8+. The percentages of cytoplasmic IgG-, IgA- and IgM-bearing cells were estimated as approximately 35, 60 and 5% of all cytoplasmic Ig-bearing cells, respectively. Deposits of immunoglobulins and third component of complement were identified on the basement membrane of the seminiferous tubules, interstitium between the tubules, vessel endothelium and degenerated germ cells in the lesion. Circulating antibodies directed against the acrosomal portion of germ cells were detected in IgG and IgM classes but not in IgA class. Inflammatory cells (including macrophages, B cells and, probably, activated T cells) in the lesion were Ia+, but Leydig cells, Sertoli cells and germ cells did not stain for Ia at all.

REVIEW ARTICLE: Patterns of Infiltration of Lymphocytes into the Testis Under Normal and Pathological Conditions in Mice

The testis is known as an immunologically privileged organ. In particular, the blood–testis barrier formed by Sertoli cells protects auto‐immunogenic spermatids from attack by the self‐immune system. We review here the micro‐status of testicular tissues in mice from the viewpoint of induction of inflammatory cell responses. Many studies have demonstrated that the testis is the most resistant to various forms of non‐autoimmune inflammation among the male reproductive organs. However, it was found that testicular inflammation of autoimmune origin is inducible by immunization with testis antigens even without an adjuvant in mice. In particular, the tubuli recti (TR) comprises specific region, where lymphocytes are attracted. Many antigen‐presenting macrophages preferentially accumulate around the TR under normal conditions. This characteristic accumulation of macrophages is an acquired phenomenon that is completed when spermatids start to differentiate in the seminiferous tubules (S). In addition, intra‐tubular lymphocytes that are very close to both germ cells and their remnants could be occasionally found in the TR, rete testis (R), epididymis (E), but not in the S, in normal mice. Although the physiological function of these penetrating lymphocytes remains unknown, we suppose that this micro‐status provides a chance for evocation of autoimmune inflammation of the TR, R and E in some pathological conditions.

Connective tissue configuration in the human liver hilar region with special reference to the liver capsule and vascular sheath

BACKGROUND/PURPOSE We carried out this study to examine the validity of the accepted dogma that: (1) the human liver capsule does not extend along the fissures for the hepatic veins; (2) the hilar vasculobiliary sheath does not connect to the liver capsule; and (3) the hilar plate is a thickening of the vasculobiliary sheath. METHODS Using cadaveric specimens, we identified composite fibers and other structures in the sheath and capsule histologically. RESULTS The liver capsule, Glissons sheath, and the sheath for the hepatic vein tributaries were characterized by a high content of thin, wavy elastic fibers. However, the hilar vasculobiliary sheath of the thick vessels and ducts did not contain elastic fibers. Along the roof of the hilar region, vaginal ductuli were identified as a chain of cross-sectional bile ducts with a relatively thick wall, because of their tortuous course with abundant small pouches budding from the surface. The ductuli were separated from the liver capsule by abundant lymphatic vessels. CONCLUSIONS The sheath for hepatic veins and Glissons sheath appear to connect to, and be continuous with, the liver capsule. During surgery and dissection, it should be borne in mind that the hilar plate is likely to be artificially developed when, without intention, surgeon bundle collagenous fibers with vaginal ductuli forming a core.