Aberrant phosphorylation of human LRH1 at serine 510 is predictable of hepatocellular carcinoma recurrence.

We previously identified the AKT-phosphorylation sites in nuclear receptors and showed that phosphorylation of S379 in mouse retinoic acid γ and S518 in human estrogen receptor α regulate their activity independently of the ligands. Since this site is conserved at S510 in human liver receptor homolog 1 (hLRH1), we developed a monoclonal antibody (mAb) that recognized the phosphorylation form of hLRH1S510 (hLRH1pS510) and verified its clinicopathological significance in hepatocellular carcinoma (HCC). We generated the anti-hLRH1pS510 mAb and assessed its selectivity. We then evaluated the hLRH1pS510 signals in 157 cases of HCC tissues by immunohistochemistry because LRH1 contributes to the pathogenesis of diverse cancers. The developed mAb specifically recognized hLRH1pS510 and worked for immunohistochemistry of formalin-fixed paraffin-embedded tissues. hLRH1pS510 was exclusively localized in the nucleus of HCC cells, but the signal intensity and positive rates varied among the subjects. According to the semi-quantification, 45 cases (34.9%) showed hLRH1pS510-high, and the remaining 112 cases (65.1%) exhibited hLRH1pS510-low. There were significant differences in the recurrence-free survival (RFS) between the two groups, and the 5-year RFS rates in the hLRH1pS510-high and hLRH1pS510-low groups were 26.5% and 46.1%, respectively. In addition, high hLRH1pS510 was significantly correlated with portal vein invasion, hepatic vein invasion, and high levels of serum alpha-fetoprotein (AFP). Furthermore, multivariable analysis revealed that hLRH1pS510-high was an independent biomarker for HCC recurrence. We conclude that aberrant phosphorylation of hLRH1S510 is a predictor of poor prognosis for HCC. The anti-hLRH1pS510 mAb could provide a powerful tool to validate the relevance of hLRH1pS510 in pathological processes such as tumor development and progression.

[A Case of Pancreatic Cancer That Developed Peritoneal Recurrence after Radical Surgery following Neoadjuvant Chemoradiation Therapy(NACRT)with Pathological Complete Response(pCR)].

A 71-year-old woman was diagnosed with a tumor in the pancreatic head on CT imaging, which was performed as a close examination of an exacerbation of diabetes mellitus. The pancreatic tumor was diagnosed as resectable pancreatic cancer, and after preoperative adjuvant chemoradiotherapy, pancreatoduodenectomy was performed as a radical surgery. There were no residual tumor cells in the resected specimen histopathologically, and the patient was judged to have a pathological complete response(pCR). Six months of postoperative adjuvant chemotherapy was administered, but peritoneal recurrence was observed at 20 months postoperatively, and the patient is currently undergoing treatment for recurrence. There have been other reports of recurrence even after pCR was achieved with preoperative treatment, so it is important to follow up carefully, keeping in mind that pancreatic cancer is a latent systemic disease.

Comprehensive analysis of gene expression of isolated pancreatic islets during pretransplant culture.

BACKGROUND: The aim of this study was to investigate the effect of pretransplant culture on the survival of pancreatic islet grafts, and to determine the biological characteristics of isolated islets during pretransplant culture. METHODS: The survival of islets from Wistar rats, transplanted to diabetic C57BL/B6 mice, was compared between fresh islets and cultured islets. A comprehensive gene expression analysis was employed to investigate biological processes during pretransplant culture, and in vitro validation studies were performed. RESULTS: Survival of cultured xenografts was significantly prolonged as compared to that of fresh islets (fresh:12.5 ± 1.9 days, 1-day cultured:16.0 ± 1.3 days (p= 0.017), 3-day cultured:17.0 ± 2.6 days (p= 0.014)). Comprehensive gene expression analysis identified significant upregulation of annotated functions associated with inflammation in cultured groups. Six proinflammatory genes, including heme oxygenase 1 (HO-1) and IL-6, were significantly upregulated during culture.　Validation studies revealed significantly higher levels of IL-6 in the supernatant of cultured islets and HO-1 in the cultured islets when compared with fresh islets. CONCLUSION: Transplantation of cultured islets induced significant but minimal prolongation of graft survival in xenogeneic combinations. Comprehensive analysis of gene expression in cultured islets showed biological processes associated with proinflammation during culture.

Ex vivo Pretreatment of Islets with Mitomycin C: Reduction in Immunogenic Potential of Islets by Suppressing Secretion of Multiple Chemotactic Factors.

Strategies to reduce the immunogenicity of pancreatic islets and to prevent the activation of proinflammatory events are essential for successful islet engraftment. Pretransplant islet culture presents an opportunity for preconditioning to improve outcomes of islet transplantation. We previously demonstrated that ex vivo mitomycin C (MMC) pretreatment and subsequent culture significantly prolonged graft survival. Fully understanding the biological process of pretreatment could result in the development of a protocol to improve the survival of islet grafts. Microarrays were employed to conduct a comprehensive analysis of genes expressed in untreated or MMC-treated rat islets that were subsequently cultured for 3 d. A bioinformatics software was used to identify biological processes that were most affected by MMC pretreatment, and validation studies, including in vivo and in vitro assay, were performed. The gene expression analysis identified significant downregulation of annotated functions associated with cellular movement and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies revealed significantly decreased levels of interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and matrix metallopeptidase 2 (MMP2) in culture supernatants of MMC-treated islets compared with controls. Moreover, we showed the suppression of leukocyte chemotactic activity of MMC-treated islets in vitro. We also showed that MMC-treated islets secreted lower levels of chemoattractants that synergistically reduced the immunogenic potential of islets. Histological and immunohistochemical analyses of the implant site revealed that infiltration of monocytes, CD3-positive T cells, and B cells was decreased in MMC-treated islets. In conclusion, the ex vivo pretreatment of islets with MMC and subsequent culture can reduce the immunogenic potential and prolong the survival of islet grafts by inducing the suppression of multiple leukocyte chemotactic factors.

Remnant left lobe torsion causing hepatic venous outflow obstruction after hepatic right lobectomy for giant hepatocellular carcinoma: report of a case.

We report the case of a 67-year-old man with remnant left liver torsion causing acute hepatic venous outflow obstruction after right hepatectomy for giant hepatocellular carcinoma, which was successfully treated with surgery. After the primary surgery, he developed significant liver dysfunction and renal failure. Doppler ultrasonography disclosed gradual reduction of hepatic perfusion. Abdominal computed tomography revealed that the swollen remnant liver was dislocated in the right subphrenic space. After surgical repositioning of the left lobe into its anatomical position, the hepatic congestion immediately disappeared, and the hemodynamic parameters improved. The falciform and round ligaments were fixed to the anterior abdominal wall to keep the remnant liver in the anatomical position. His postoperative course was uneventful. Doppler ultrasonography was useful to assess hepatic perfusion for screening of acute hepatic venous outflow obstruction and abdominal computed tomography is definitive for diagnosis. Fixation of remnant liver may be effective for preventing hepatic venous outflow obstruction after right hepatectomy, particularly for giant tumor.

[A case of two re-resections for peritoneal recurrence of pancreatic cancer].

A 63-year-old man underwent subtotal stomach-preserving pancreaticoduodenectomy and intraoperative radiation therapy for pancreatic head cancer (Stage IVa, JPS). Subsequently, he received adjuvant chemotherapy including gemcitabine (GEM). A peritoneal recurrence appeared adjacent to the right colon and the lower pole of the right kidney. He received chemotherapy including S-1 and GEM+S-1. Since the recurrent lesions were localized, he underwent resection for the recurrence lesions 2 years and 9 months after surgery. The resection was R1, and re-recurrence appeared in the lower pole of the right kidney. After chemotherapy and chemoradiotherapy, he underwent re-resection for the recurrence 4 years and 3 months after the first surgery. Three months after re-resection, multiple liver metastases appeared in the bilateral lobes. Reoperation can be one of the multidisciplinary management options for local recurrence of pancreatic cancer under specific conditions.

Mitomycin C treatment significantly reduces central damage of islets in culture.

OBJECTIVES: We recently reported that mitomycin C (MMC) treatment and subsequent culture of islets significantly prolongs graft survival in allotransplantation and xenotransplantation models. The present study was performed to determine the changes in morphology and signal transduction in pancreatic islets after MMC treatment. METHODS: Freshly isolated rat islets were treated with 10 µg/mL MMC for 30 minutes and then cultured for up to 3 days. The samples were processed for immunohistologic studies and electron microscopic examination at various times after treatment. A DNA fragmentation assay was performed to detect apoptotic cell death. Western blotting was performed to determine the effects of MMC on signal transduction. RESULTS: As early as 4 hours after culture, the islets showed central damage; most cells were necrotic and stained with anti-high mobility group box 1 antibody, and a few were apoptotic. The ratio of the damaged area to the whole area was significantly decreased after MMC treatment. Western blotting showed that MMC treatment increased the levels of activated forms of p53 and p21, whereas levels of the activated forms of Akt and caspase-3 were unchanged. CONCLUSIONS: Mitomycin C treatment protects islets from the progression of central damage during culture. The p53-p21 pathway might be involved in these effects. ABBREVIATIONS: MMC - mitomycin C, HMGB1 - high mobility group box 1.

Improved islet yield and function by use of a chloride channel blocker during collagenase digestion.

BACKGROUND: Protection of pancreatic islets during isolation procedures is mandatory for successful islet transplantation. Chloride channel inhibition has been reported to prevent cell death induced by various stimuli. We examined the effects of the chloride channel blocker, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt (DIDS) and extracellular Cl(-)-free conditions on islet isolation outcomes. METHODS: Experimental groups were created based on the collagenase solutions used for Wistar rat islet isolation: control group, Hanks' balanced salt solution; DIDS group, 200 µM DIDS; and Cl(-)-free group, sodium gluconate substituted for sodium chloride. We determined whether collagenase digestion induced the death of islet cells through Cl(-) influx into the cells. We then assessed islet yield and the viability of isolated islets. RESULTS: We observed an increase in intracellular Cl(-) concentration under collagenase digestion conditions using a Cl(-)-sensitive fluorescent dye and subsequent rupture of islet cells. Consequently, islet yields were significantly higher in the DIDS and Cl(-)-free groups than in the control group, and islet morphology of the former groups was preserved. Of streptozotocin-induced diabetic severe combined immunodeficiency mice transplanted with a marginal dose of islets, all seven mice in the DIDS group and six of the seven mice in the Cl(-)-free group became normoglycemic, compared with two of seven mice in the control group (control vs. DIDS, P=0.010; control vs. Cl(-) free, P=0.051). CONCLUSION: Our results indicate that DIDS inhibition of Cl(-) influx into islets protects islets during digestion procedures, offering a new strategy for the improvement of islet isolation outcomes.