Noriyoshi Sueyoshi

Death receptor 5 mediated-apoptosis contributes to cholestatic liver disease

Chronic cholestasis often results in premature death from liver failure with fibrosis; however, the molecular mechanisms contributing to biliary cirrhosis are not demonstrated. In this article, we show that the death signal mediated by TNF-related apoptosis-inducing ligand (TRAIL) receptor 2/death receptor 5 (DR5) may be a key regulator of cholestatic liver injury. Agonistic anti-DR5 monoclonal antibody treatment triggered cholangiocyte apoptosis, and subsequently induced cholangitis and cholestatic liver injury in a mouse strain-specific manner. TRAIL- or DR5-deficient mice were relatively resistant to common bile duct ligation-induced cholestasis, and common bile duct ligation augmented DR5 expression on cholangiocytes, sensitizing mice to DR5-mediated cholangitis. Notably, anti-DR5 monoclonal antibody-induced cholangitis exhibited the typical histological appearance, reminiscent of human primary sclerosing cholangitis. Human cholangiocytes constitutively expressed DR5, and TRAIL expression and apoptosis were significantly elevated in cholangiocytes of human primary sclerosing cholangitis and primary biliary cirrhosis patients. Thus, TRAIL/DR5-mediated apoptosis may substantially contribute to chronic cholestatic disease, particularly primary sclerosing cholangitis.

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.

Apoptosis as a Possible Cause of Wall Thinning in End-Stage Hypertrophic Cardiomyopathy

A 15-year-old boy with hypertrophic cardiomyopathy died of congestive heart failure with progressive left ventricular wall thinning with poor systolic function. Microscopic examination revealed patchy fibrosis in the ventricular myocardium with wall thinning, and immunohistochemical evaluation of apoptosis showed apoptotic cells and bodies in the destroyed myocytes along the border between the fibrotic area and myofibril.

Are stable postoperative biliary atresia patients really stable

Abstract Transforming growth factor-beta 1 (TGF β-1) is an important mediator of liver-cell proliferation and replication that is implicated in hepatic fibrosis (HF). Hepatic stellate cells (HSC) are activated by TGF β-1 and are the main precursor cells involved in fibrogenesis. The correlation between serum TGF β-1, activated HSC in liver-biopsy specimens, and liver biochemistry was investigated to determine the value of TGF β-1 as an indicator of clinical status in postoperative biliary atresia (BA) patients. Thirty-two postoperative BA patients (mean age 11.2 ± 2.8 years) and 13 normal controls (mean age 10.3 ± 3.7 years) were studied. Based on average liver function test (LFT) results over a 3-month period immediately prior to this study, the BA patients were divided into group I (anicteric, normal LFT; n = 10); group II (anicteric, elevated liver transaminases; n = 12), and group III (jaundiced end-stage liver fibrosis awaiting liver transplantation; n = 10). Serum TGF β-1 was determined using ELISA. Liver-biopsy specimens were examined with antibody against TGF β-1 and α-smooth muscle actin (SMA) antibody for detection of activated HSC. Serum TGF β-1 was significantly higher in groups I (11.4 ± 3.7 ng/ml; P < 0.01) and II (23.3 ± 11.3 ng/ml; P < 0.001) than in group III (3.0 ± 1.5 ng/ml) and controls (4.5 ± 2.5 ng/ml) despite normal LFT in group I. The 3 subjects with the highest serum TGF β-1 in group II had bile lakes. Biopsies from groups I and II were strongly positive for TGF β-1 in hepatocytes and Kupffer cells and for activated HSC detected by SMA compared with group III and controls. Because serum TGF β-1 and activated HSC are only present during active fibrosis, we conclude that there is progressive fibrogenesis even in seemingly normal postoperative BA patients. In particular, bile lakes should be regarded as a key sign of progressive HF, the presence of which should be regarded with extreme caution. We suggest that serum TGF β-1 could be used as an accurate indicator of progressive fibrogenesis in postoperative BA patients.

Mesenchymal stem cell transplantation to the mouse cochlea as a treatment for childhood sensorineural hearing loss

OBJECTIVE There is no treatment established for congenital sensorineural hearing loss because the majority of the cases are hereditary. Although congenital sensorineural hearing loss is thought to be hereditary, this hearing loss occur postnatally. We hypothesized that the transplantation of MSCs (mesenchymal stem cells) to the cochlea would be an effective therapy for stopping or delaying the progression of sensorineural hearing loss in childhood. METHODS Cultured mouse MSCs were labeled with EGFP (enhanced green fluorescence protein) using retroviruses. EGFP-MSCs were transplanted into the posterior semicircular canal of mice at 2-3 weeks (young group) and 24-26 weeks (adult group) of age by a novel perilymphatic perfusion technique. Engraftment of MSCs was evaluated immunohistologically at 1 week and 2 weeks after transplantation. RESULTS In young mice, migrated MSCs were detected in the cochlea tissue by immunofluorescence for EGFP and by immunohistochemistry for fibronectin. The differentiation of migrated MSCs into fibrocyte-like cells was demonstrated by immunofluorescence for connexin 26. There were no adverse effects on auditory function by MSC transplantation, and the auditory brain stem responses threshold did not significantly shift after surgery. In contrast, neither MSC migration nor differentiation was detected in the adult mice canal after MSC transplantation. CONCLUSION The bone marrow derived MSCs were successfully transplanted into the cochlea of young mice by the perilymphatic perfusion technique and were further differentiated into fibrocyte-like cells without any adverse effects on auditory function.

Lack of C-KIT+ mast cells and the development of idiopathic gastric perforation in neonates.

BACKGROUND/PURPOSE The proto-oncogene c-kit encodes a receptor tyrosine kinase C-KIT. W/Wv mice, which are devoid of C-KIT+ mast cells as a result of mutations in the c-kit gene, develop spontaneous gastric ulceration or perforation after day 7 of life at a high frequency, whereas normal litter mates do not. The authors hypothesized that a lack of C-KIT+ mast cells may be implicated in the development of idiopathic gastric perforation (GP) in neonates. METHODS Postmortem gastric wall specimens were taken from neonates who died of GP (idiopathic, n = 6; secondary, n = 4), and other causes (controls, n = 6). Specimens were taken at random from various sites in the stomach and labeled with antibody to C-KIT. The number of C-KIT+ mast cells from five random fields per specimen were compared under light microscopy (200x). RESULTS Overall, the number of C-KIT+ mast cells was significantly lower in gastric wall specimens from cases of idiopathic GP when compared with controls or cases of secondary GP irrespective of the sites of sampling (P<.01, analysis of variance test) with the distribution of cells being uniform and unique for each stomach. CONCLUSION A lack of C-KIT+ mast cells may underlie the development of idiopathic GP in neonates.

Necrotizing enterocolitis and C-KIT

BACKGROUND/PURPOSE In the gut, C-KIT is important for immune system homeostasis, and C-KIT+ cells are known to increase during inflammation. Recently the authors identified that spontaneous intestinal mucosal erosion develops in C-KIT-depleted W/Wv mice after day 14 of life at a high frequency, whereas genotypically normal litter mates do not. The authors hypothesized that a lack of C-KIT may be implicated in the development of necrotizing enterocolitis (NEC). METHODS Bowel specimens were taken during surgery or postmortem from nine cases of NEC (mean gestational age, 32.0 weeks), six age-matched cases of enteritis, and 10 age-matched controls. Specimens were formalin fixed, paraffin embedded, and labeled with antibody to C-KIT. The number of C-KIT+ cells from five random fields per specimen were compared under light microscopy (200x). Results were expressed as the mean +/- SD and compared using the analysis of variance (ANOVA) test. RESULTS In enteritis, the number of C-KIT+ cells in the lamina propria and submucosa was significantly higher than in controls (P<.01) indicative of their involvement in inflammation. However, in NEC, the number of C-KIT+ cells in the lamina propria and submucosa was significantly lower than in controls (P<.05) despite histological evidence of inflammation. CONCLUSION A lack of C-KIT+ cells may exert a causal influence on the development of NEC.

Prediction of radiosensitivity using phosphorylation of histone H2AX and apoptosis in human tumor cell lines.

Abstract Purpose: We examined the relationship between radiosensitivity, histone H2AX (γH2AX) phosphorylation, and apoptosis to develop a new predictive assay for radiosensitivity. Materials and methods: Seven human tumor cell lines, including one fibrosarcoma (HT1080), four oesophageal carcinomas (TE-9, KYSE30, KYSE150, and KYSE220), and two breast carcinomas (HCC70, and ZR75-1) were used. Cellular radiosensitivity was assessed using a standard colony-forming assay. To measure the frequency of γH2AX foci, we counted the number of foci per cell under fluorescence microscopy following immunofluorescence staining. DNA content was determined by a flow cytometric assay. To assess the frequency of apoptosis, we enumerated apoptotic cells by fluorescence microscopy 24 hours after irradiation. Results: All seven cell lines showed dose (0–9 Gy)-dependent increases in the number of γH2AX foci per cell 24 h after irradiation. When both the frequency of γH2AX foci normalized by DNA content and the frequency of apoptosis were used, a better correlation was observed between the actual cell survivals and the predicted ones. Conclusions: Our study shows that the number of γH2AX foci after normalization of the DNA content and apoptotic cell frequency can be used as a new predictive assay for cell survival.

Transplantation of infantile bladder in rats: an alternative procedure for bladder augmentation.

Background. Our purpose was to evaluate whether bladder transplantation (BTx) can be used for bladder augmentation (BA). Methods. Bladders from infantile Brown-Norway rats (less than 21 days old) were excised and each transplanted into a pouch created in the distal omentum of a 6-week-old Lewis rat (fully allogeneic BTx). No immunosuppressant was used in group I (n=12). Intramuscular FK506 was used daily from the day of BTx in group II (n=16; 0.2 mg/kg), group III (n=22; 0.6 mg/kg), and group IV (n=16; 1.2 mg/kg) until harvesting 3, 4, 5, or 6 weeks after BTx. FK506 was used for only 2 weeks in group V (n=12; 0.6 mg/kg/day) and group VI (n=12; 1.2 mg/kg/day). Syngeneic bladder transplants acted as controls (n=16). Hematoxylin and eosin staining was used to examine all grafts. In six rats from group III, BA was performed by anastomosing the graft to the recipient bladder 10 days after BTx. Results. Each successfully transplanted graft appeared macroscopically as a thin-walled cyst. Rejection was seen in all grafts from groups I, II, V, and VI, and was minimal or absent in groups III and IV. On medium to long-term follow-up the only side effect of FK506 observed was reduced weight gain. Graft survival in the control group was 100%. BA was successful in all six cases, and the mucosa was normal throughout each augmented bladder. Conclusions. This is the first report of the successful transplantation of infantile tissue without vascular anastomosis. Because of the efficient, safe immunosuppression possible with FK506, our BTx technique could find clinical application for creating viable vesical tissue that could be used for BA.

Long-term outcome of bladder augmentation using living-related partial bladder transplantation in rats

Long-term histopathologic changes after bladder augmentation (BA) in rats using living-related partial bladder transplantation (LPBTx) or conventional ileocystoplasty (ICP) were compared. In this study, BA (n = 37), LPBTx (n = 18), and ICP (n = 19) were performed in 16-wk-old Lewis rats. Five donors and seven nontransplanted normal Lewis rats (controls) were also studied. Rats that survived >10 mo after BA were killed after blood biochemistry and neobladder imaging. Harvested bladders were examined with hematoxylin and eosin and proliferating cell nuclear antigen (PCNA). When the rats were killed, there were 16 rats in the LPBTx group and 12 rats in the ICP group; ICP rats were significantly smaller than LPBTx rats (p < 0.05). Mean duration of follow-up for the LPBTX group was 17.3 mo, for the ICP group was 13.7 mo, for the donor group was 16.1 mo, and for the control group was 19.7 mo. Mean serum pH in the LPBTx group was 7.41 ± 0.78 and in the ICP group was 7.25 ± 0.38. Mean base excess in the ICP group was significantly lower than in the LPBTx group (p < 0.05). Incidence of bladder calculi in the LPBTx group (6.3%) was significantly lower than in the ICP group (33.3%; p < 0.05). There was no dysplasia/malignancy/increase in PCNA in the LPBTx group. PCNA increased in the ICP group, compared with controls (p < 0.05); two (16.7%) of 12 of ICP rats had dysplasia with mitosis. Bladder capacity increased in LPBTx and ICP compared with controls (both p < 0.05). We hope to show that BA using LPBTx may result in a neobladder with fewer complications than BA using ICP; LPBTx may also decrease the risk for malignancy.