Munekazu Naito

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.

Vitamin K2 induces autophagy and apoptosis simultaneously in leukemia cells

Vitamin K2 (menaquinone-4: VK2) is a potent inducer for apoptosis in leukemia cells in vitro. HL-60bcl-2 cells, which are derived from a stable transfectant clone of the human bcl-2 gene into the HL-60 leukemia cell line, show 5-fold greater expression of the Bcl-2 protein compared with HL-60neo cells, a control clone transfected with vector alone. VK2 induces apoptosis in HL-60neo cells, whereas HL-60bcl-2 cells are resistant to apoptosis induction by VK2 but show inhibition of cell growth along with an increase of cytoplasmic vacuoles during exposure to VK2. Electron microscopy revealed formation of autophagosomes and autolysosomes in HL-60bcl-2 cells after exposure to VK2. An increase of acid vesicular organelles (AVOs) detected by acridine orange staining for lysosomes as well as conversion of LC3B-I into LC3B-II by immunonoblotting and an increased punctuated pattern of cytoplasmic LC3B by fluorescent immunostaining all supported induction of enhanced autophagy in response to VK2 in HL-60bcl-2 cells. However, during shorter exposure to VK2, the formation of autophagosomes was also prominent in HL-60neo cells although nuclear chromatin condensations and nuclear fragments were also observed at the same time. These findings indicated the mixed morphologic features of apoptosis and autophagy. Inhibition of autophagy by either addition of 3-methyladenine, siRNA for Atg7, or Tet-off Atg5 system all resulted in attenuation of VK2-incuded cell death, indicating autophagy-mediated cell death in response to VK2. These data demonstrate that autophagy and apoptosis can be simultaneously induced by VK2. However, autophagy becomes prominent when the cells are protected from rapid apoptotic death by a high expression level of Bcl-2.

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.

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.

Absence of the celiac trunk: case report and review of the literature.

The authors report a rare variation of the absence of the celiac trunk in a Japanese cadaver, with the left gastric, splenic, common hepatic, and superior mesenteric arteries arising independently from the abdominal aorta in the routine dissection of a 95‐year‐old Japanese male cadaver. The incidence and developmental and clinical significance of this variation is discussed with a detailed review of the literature. Knowledge of such case has important clinical significance in an abdominal operation or invasive arterial procedure, that is, Appleby procedure and liver transplantation, laparoscopic surgery, and radiological procedures in the upper abdomen. Clin. Anat. 21:283–286, 2008.

Experimental autoimmune orchitis as a model of immunological male infertility

Clinically, 60–75% of male infertility cases are categorized as idiopathic spermatogenic disturbance. In previous studies of this condition, lymphocytic infiltration and immune deposits were present in several testis biopsy specimens, indicating that inflammatory or immunological factors contribute to the occurrence of the lesions. However, there is currently little evidence regarding immunological infertility in men. Previously, we established an immunological infertility model, experimental autoimmune orchitis (EAO), that can be induced in mice by two subcutaneous injections of viable syngeneic testicular germ cells without the use of any adjuvant. In this EAO model, lymphocytes surround the tubuli recti and then induce spermatogenic disturbance. In addition, after the active inflammation stage of this model, the seminiferous epithelium is damaged irreversibly, resembling the histopathology of human male idiopathic spermatogenic disturbance. In the majority of patients with testicular autoimmunity, there is a chronic and asymptomatic development of the inflammatory reaction. Therefore, this disease is very difficult to diagnose at the ongoing stage, and it is possible that the histopathology of idiopathic spermatogenic disturbance in the clinic is reported at the post-active inflammation stage of autoimmune orchitis. In this review, the histopathology of EAO before and after inflammation is discussed, comparing it with human orchitis.

A rare case of inferior mesenteric artery arising from the superior mesenteric artery, with a review of the review of the literature

Anatomical variations of the inferior mesenteric artery (IMA) are uncommon. Generally, the IMA is very stable, arises directly from the abdominal aorta at the level of the third lumbar vertebra. We describe here an extremely rare case in which the IMA arose from the superior mesenteric artery. The findings were made during routine dissection of the cadaver of a 79-year-old Japanese man. This present case is the ninth report of this variation and was associated with gastrophrenic trunk, hepatosplenic trunk, hypoplastic spleen and accessory spleen. Clinically, cases like this highlight the importance of knowing the IMA anatomy and the possibility of its numerous variations in surgical procedures such as right hemicolectomy, resection of the transverse colon, left hemicolectomy, sigmoidectomy, and en bloc resection of the head of the pancreas and the superior mesenteric vessels. The developmental significance of this variation is also discussed with a detailed review of the literature.

Clarithromycin enhances bortezomib-induced cytotoxicity via endoplasmic reticulum stress-mediated CHOP (GADD153) induction and autophagy in breast cancer cells

The specific 26S proteasome inhibitor, bortezomib (BZ) potently induces apoptosis as well as autophagy in metastatic breast cancer cell lines such as MDA-MB-231 and MDA-MB-468. The combined treatment of clarithromycin (CAM) and BZ significantly enhances cytotoxicity in these cell lines. Although treatment with up to 100 μg/ml CAM alone had little effect on cell growth inhibition, the accumulation of autophagosomes and p62 was observed after treatment with 25 μg/ml CAM. This result indicated that CAM blocked autophagy flux. However, the combined treatment of BZ and CAM resulted in more pronounced autophagy induction, as assessed by increased expression ratios of LC3B-II to LC3B-I and clearance of intracellular p62, than treatment with BZ alone. This combination further enhanced induction of the pro-apoptotic transcription factor CHOP (CADD153) and the chaperone protein GRP78. Knockdown of CHOP by siRNA attenuated the death-promoting effect of BZ in MDA-MB-231 cells. A wild-type murine embryonic fibroblast (MEF) cell line also exhibited enhanced BZ-induced cytotoxicity with the addition of CAM, whereas a Chop knockout MEF cell line completely abolished this enhancement and exhibited resistance to BZ treatment. These data suggest that endoplasmic reticulum (ER)-stress mediated CHOP induction is involved in pronounced cytotoxicity by combining these reagents. Simultaneously targeting two major intracellular protein degradation pathways such as the ubiquitin-proteasome system by BZ and the autophagy-lysosome pathway by CAM may improve the therapeutic outcome in breast cancer patients via ER-stress mediated apoptosis.

Caput epididymitis but not orchitis was induced by vasectomy in a murine model of experimental autoimmune orchitis

Immunization of mice with viable syngeneic testicular germ cells (TGC) alone can induce autoimmune responses against autoantigens of both round and elongating spermatids, resulting in the development of experimental autoimmune orchitis (EAO). Histological lesions in this EAO model without an adjuvant are characterized by lymphocytic infiltration into the testes, spermatogenic disturbance, and a complete lack of epididymitis. In this study, we investigated the effects of vasectomy (Vx) on TGC-induced EAO expecting that Vx augments the severity of testicular inflammation in A/J mice. The results showed that mice receiving Vx alone exhibited no significant inflammatory cell response in either the testes or epididymides, and mice receiving shamVx+TGC immunization had EAO with no epididymitis. In sharp contrast, no EAO was found in the testes of any mice receiving Vx+TGC immunization. Instead, caput epididymitis involving CD4+T cells, CD8+T cells, B cells, and macrophages were induced in them with striking elevation of the tissue levels of both IL6 and IL10 mRNA. Furthermore, serum autoantibodies induced by shamVx+TGC immunization were reactive with both round (immature) and elongating (mature) spermatids; however, those induced by Vx+TGC immunization were specific to acrosomes of mature spermatids and spermatozoa. These unexpected results indicate that Vx may induce the mode by which autoreactive lymphocytes gain access to TGC autoantigens in the epididymides, leading to autoimmune responses against the autoantigens of mature rather than immature spermatids.

History and future of human cadaver preservation for surgical training: from formalin to saturated salt solution method

Traditionally, surgical training meant on-the-job training with live patients in an operating room. However, due to advancing surgical techniques, such as minimally invasive surgery, and increasing safety demands during procedures, human cadavers have been used for surgical training. When considering the use of human cadavers for surgical training, one of the most important factors is their preservation. In this review, we summarize four preservation methods: fresh-frozen cadaver, formalin, Thiel’s, and saturated salt solution methods. Fresh-frozen cadaver is currently the model that is closest to reality, but it also presents myriad problems, including the requirement of freezers for storage, limited work time because of rapid putrefaction, and risk of infection. Formalin is still used ubiquitously due to its low cost and wide availability, but it is not ideal because formaldehyde has an adverse health effect and formalin-embalmed cadavers do not exhibit many of the qualities of living organs. Thiel’s method results in soft and flexible cadavers with almost natural colors, and Thiel-embalmed cadavers have been appraised widely in various medical disciplines. However, Thiel’s method is relatively expensive and technically complicated. In addition, Thiel-embalmed cadavers have a limited dissection time. The saturated salt solution method is simple, carries a low risk of infection, and is relatively low cost. Although more research is needed, this method seems to be sufficiently useful for surgical training and has noteworthy features that expand the capability of clinical training. The saturated salt solution method will contribute to a wider use of cadavers for surgical training.