Intraneural fibrosis within ilioinguinal nerve in inguinal hernia patients with preoperative pain: it's the sign of irreversible nerve injury, isn't it?

PURPOSE: Preoperative pain is known as one of the most powerful risk factors for chronic postoperative inguinal pain (CPIP), while its pathogenesis has not been fully elucidated. The aim of the present study was to evaluate patients with preoperative pain from the pathological perspective and discuss the potential pathogenesis of CPIP in those patients. METHODS: This was a single-institutional retrospective study. The study population was inguinal hernia patients with preoperative pain who underwent open anterior hernia repair for primary inguinal hernia with pragmatic ilioinguinal neurectomy during surgery between March 2021 and March 2023. The primary and secondary outcomes were proportion of collagen deposition and mucus accumulation within ilioinguinal nerve in those patients, respectively, which were evaluated histologically using Image J software. RESULTS: Forty patients were evaluated. Median value of proportion of intraneural collagen deposition was 38.3% (27.7-95.9). These values were positively correlated with the duration of pain (r2=0.468, P<0.001). Median value of proportion of mucus accumulation in ilioinguinal nerve was 50.1% (0-82.0). These values had no correlation with any clinicopathological variables. CONCLUSIONS: In the present study population, all patients with preoperative pain had intraneural fibrosis within ilioinguinal nerve, and its degree had a positive correlation with the pain duration.

A pathological perspective to painful inguinal hernia: Report of two cases.

INTRODUCTION: Preoperative inguinal pain (painful inguinal hernia) is a well-known factor associated with chronic postoperative inguinal pain (CPIP). However, it remains unclear what preventive measures should be taken in such patients. CASE PRESENTATION: We report two patients with painful inguinal hernia who underwent pragmatic ilioinguinal nerve neurectomy during open anterior repair. The nerve was compressed by bulky spermatic cord lipoma in case 1 and by the hernia sac presenting over a few decades in case 2. Hematoxylin and eosin staining of the resected nerves revealed mucoid degeneration. Toluidine blue staining of resin-embedded nerve sections demonstrated that fully-myelinated axons had significantly decreased in case 1 and almost disappeared in case 2, indicating the development of massive demyelination of the ilioinguinal nerve in both cases. DISCUSSION: In cases where the injured nerve is left in situ, CPIP may occur since demyelinating neuropathy sometimes becomes irreversible. CONCLUSION: Planned nerve resection via open anterior inguinal hernia repair may be an option to prevent CPIP in patients with painful inguinal hernia.

Mesh shrinkage is the potential pathogenesis of chronic somatic pain following transabdominal preperitoneal repair: Report of two cases.

Although mesh-related pain, termed "somatic pain," is a well-known pain syndrome following Lichtenstein repair, few reports are available on somatic pain following transabdominal preperitoneal repair (TAPP) and its pathogenesis remains unclear. We report on two patients with refractory somatic chronic pain following TAPP. In the present two cases, both mesh fixation with rigid permanent metal tackers and mesh shrinkage resulting in contractile forces on the groin musculature could be considered as potential mechanisms in the etiology of chronic somatic pain following TAPP. The lessons learned from these two cases are: (a) mesh shrinkage resulting in contractile forces on the groin musculature could be considered as potential mechanisms in the etiology of chronic somatic pain following TAPP; (b) partial mesh removal would be an effective alternative to total mesh removal in those patients for remedial surgery.

Novel hybrid three-dimensional artificial liver using human induced pluripotent stem cells and a rat decellularized liver scaffold.

INTRODUCTION: Liver transplantation is currently the only curative therapy for end-stage liver failure; however, establishment of alternative treatments is required owing to the serious donor organ shortage. Here, we propose a novel model of hybrid three-dimensional artificial livers using both human induced pluripotent stem cells (hiPSCs) and a rat decellularized liver serving as a scaffold. METHODS: Rat liver harvesting and decellularization were performed as reported in our previous studies. The decellularized liver scaffold was recellularized with hiPSC-derived hepatocyte-like cells (hiPSC-HLCs) through the biliary duct. The recellularized liver graft was continuously perfused with the culture medium using a pump at a flow rate of 0.5 mL/min in a standard CO2 (5%) cell incubator at 37 °C. RESULTS: After 48 h of continuous perfusion culture, the hiPSC-HLCs of the recellularized liver distributed into the parenchymal space. Furthermore, the recellularized liver expressed the albumin (ALB) and CYP3A4 genes, and secreted human ALB into the culture medium. CONCLUSION: Novel hybrid artificial livers using hiPSCs and rat decellularized liver scaffolds were successfully generated, which possessed human hepatic functions.

CD90 expression in human intrahepatic cholangiocarcinoma is associated with lymph node metastasis and poor prognosis.

BACKGROUND AND OBJECTIVES: Intrahepatic cholangiocarcinoma (ICC) is the second most common primary liver cancer. However, its prognosis remains poor. Expression of cluster of differentiation 90 (CD90) has been identified as an indicator of poor prognosis in many cancers. Here, we examined the importance of CD90 expression in ICC. METHODS: We performed immunohistological assays for CD90 in human ICC surgical specimens and assessed its relationship with clinicopathological findings and prognosis. Moreover, we analyzed the characteristics of CD90+/- cells, mainly with respect to metastatic potential, using human ICC cell lines. RESULTS: CD90 expression was significantly associated with lymph node metastasis and was revealed to be an independent prognostic factor. The CD90+ cells present in ICC specimens did not appear to be cancer-associated fibroblasts, as they did not express α-smooth muscle actin. In vitro, CD90 + cells exhibited greater migratory ability and higher expression of epithelial-mesenchymal transition (EMT)-related genes, including CXCR4 and MMP7, than CD90- cells. Wnt/ß-catenin signaling pathway activation was also heightened in CD90+ cells. In such cells, EMT appeared to be induced by CXCR4 and MMP7 expression through activation of Wnt/ß-catenin signaling. CONCLUSION: CD90+ cells demonstrate high metastatic potential owing to Wnt/ß-catenin signaling activation and are associated with poor prognosis in ICC.

Establishment of practical recellularized liver graft for blood perfusion using primary rat hepatocytes and liver sinusoidal endothelial cells.

Tissue decellularization produces a three-dimensional scaffold that can be used to fabricate functional liver grafts following recellularization. Inappropriate cell distribution and clotting during blood perfusion hinder the practical use of recellularized livers. Here we aimed to establish a seeding method for the optimal distribution of parenchymal and endothelial cells, and to evaluate the effect of liver sinusoidal endothelial cells (LSECs) in the decellularized liver. Primary rat hepatocytes and LSECs were seeded into decellularized whole-liver scaffolds via the biliary duct and portal vein, respectively. Biliary duct seeding provided appropriate hepatocyte distribution into the parenchymal space, and portal vein-seeded LSECs simultaneously lined the portal lumen, thereby maintaining function and morphology. Hepatocytes co-seeded with LSECs retained their function compared with those seeded alone. Platelet deposition was significantly decreased and hepatocyte viability was maintained in the co-seeded group after extracorporeal blood perfusion. In conclusion, our seeding method provided optimal cell distribution into the parenchyma and vasculature according to the three-dimensional structure of the decellularized liver. LSECs maintained hepatic function, and supported hepatocyte viability under blood perfusion in the engineered liver graft owing to their antithrombogenicity. This recellularization procedure could help produce practical liver grafts with blood perfusion.

Identification of keratin 19-positive cancer stem cells associating human hepatocellular carcinoma using CYFRA 21-1.

The current lack of an easily measurable surrogate marker of cancer stem cells (CSCs) prevents the clinical application of CSCs for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker associated with transforming growth factor beta (TGFß)/Smad signaling, and that K19+ HCC-CSCs could be a new therapeutic target of TGFß receptor 1 inhibitor LY2157299. In this study, we examined whether K19+ HCC-CSCs can be tracked using cytokeratin 19 fragment CYFRA 21-1. In 147 HCC patients who underwent curative resection and evaluated K19 expression by immunohistochemistry, preoperative serum CYFRA 21-1 levels were significantly higher in K19+ patients than in K19- patients (P < 0.01). Receiver operating characteristic analyses revealed that serum CYFRA 21-1 was the statistically significant and the most sensitive predictor of tumor K19 expression among preoperative laboratory test values (P < 0.001). In HCC cells encoding with a K19 promoter-driven enhanced green fluorescent protein, fluorescence-activated cell sorting (FACS)-isolated K19+ cells displayed significantly higher levels of supernatant CYFRA 21-1 than K19- cells (P < 0.01). Gain/loss of K19 function experiments confirmed that CYFRA 21-1 levels were regulated by K19 function in HCC cells. Furthermore, CYFRA 21-1 levels reflected the treatment efficacy of LY2157299 in K19+ cells. In conclusion, CYFRA 21-1 can be used to predict K19 expression in HCC, and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC-CSCs.

A novel three-dimensional culture system maintaining the physiological extracellular matrix of fibrotic model livers accelerates progression of hepatocellular carcinoma cells.

Liver fibrosis is characterized by the progressive accumulation of extracellular matrix (ECM) and is a strong predictor of hepatocellular carcinoma (HCC) development and progression. However, the effect of ECM in fibrotic livers on HCC cells is poorly understood. The aims of this study were to create a new culture system that retained the natural ECM of fibrotic model livers and to establish whether natural ECM regulated the characteristics of HCC cells. Using an organ decellularization technique, we created a new culture system that preserved the tissue-specific ECM of fibrotic model livers from CCl4-treated rats. The content of ECM in fibrotic model liver scaffolds was increased and the ECM microstructure was distorted. Quantitative polymerase chain reaction and immunofluorescence assays of HCC cells cultured in fibrotic model liver scaffolds for 7 days showed an epithelial-mesenchymal transition phenotype. Moreover, the ECM of fibrotic model livers promoted proliferation and chemoresistance of HCC cells. These results showed a novel effect of natural ECM in fibrotic model livers on the malignant behaviour of HCC cells. This new culture system will be useful for both understanding the cell biology of fibrotic livers and developing novel anti-cancer drugs.

Generation of non-viral, transgene-free hepatocyte like cells with piggyBac transposon.

Somatic cells can be reprogrammed to induced hepatocyte-like cells (iHeps) by overexpressing certain defined factors in direct reprogramming techniques. Of the various methods to deliver genes into cells, typically used genome-integrating viral vectors are associated with integration-related adverse events such as mutagenesis, whereas non-integrating viral vectors have low efficiency, making viral vectors unsuitable for clinical application. Therefore, we focused on developing a transposon system to establish a non-viral reprogramming method. Transposons are unique DNA elements that can be integrated into and removed from chromosomes. PiggyBac, a type of transposon, has high transduction efficiency and cargo capacity, and the integrated transgene can be precisely excised in the presence of transposase. This feature enables the piggyBac vector to achieve efficient transgene expression and a transgene-free state, thus making it a promising method for cell reprogramming. Here, we attempted to utilize the piggyBac transposon system to generate iHeps by integrating a transgene consisting of Hnf4a and Foxa3, and successfully obtained functional iHeps. We then demonstrated removal of the transgene to obtain transgene-free iHeps, which still maintained hepatocyte functions. This non-viral, transgene-free reprogramming method using the piggyBac vector may facilitate clinical applications of iHeps in upcoming cell therapy.

Identification of Keratin 19-Positive Cancer Stem Cells Associating Human Hepatocellular Carcinoma Using 18F-Fluorodeoxyglucose Positron Emission Tomography.

Purpose: The current lack of tools for easy assessment of cancer stem cells (CSC) prevents the development of therapeutic strategies for hepatocellular carcinoma (HCC). We previously reported that keratin 19 (K19) is a novel HCC-CSC marker and that PET with 18F-fluorodeoxyglucose (18F-FDG) is an effective method for predicting postoperative outcome in hepatocellular carcinoma. Herein, we examined whether K19+ HCC-CSCs can be tracked using 18F-FDG-PET.Experimental Design: K19 and glucose transporter-1 (GLUT1) expression was evaluated by IHC in 98 hepatocellular carcinoma patients who underwent 18F-FDG-PET scans before primary tumor resection. Standardized uptake values (SUV) for primary tumors and tumor-to-nontumor SUV ratios (TNR) were calculated using FDG accumulation levels, and values were compared among K19+/K19- patients. Using hepatocellular carcinoma cell lines encoding with a K19 promoter-driven enhanced GFP, 18F-FDG uptake and GLUT1 expression were examined in FACS-isolated K19+/K19- cells.Results: In hepatocellular carcinoma patients, K19 expression was significantly correlated with GLUT1 expression and FDG accumulation. ROC analyses revealed that among preoperative clinical factors, TNR was the most sensitive indicator of K19 expression in hepatocellular carcinoma tumors. In hepatocellular carcinoma cells, FACS-isolated K19+ cells displayed significantly higher 18F-FDG uptake than K19- cells. Moreover, gain/loss-of-function experiments confirmed that K19 regulates 18F-FDG uptake through TGFß/Smad signaling, including Sp1 and its downstream target GLUT1.Conclusions:18F-FDG-PET can be used to predict K19 expression in hepatocellular carcinoma and should thereby aid in the development of novel therapeutic strategies targeting K19+ HCC-CSCs. Clin Cancer Res; 23(6); 1450-60. ©2016 AACR.

Efficient recellularisation of decellularised whole-liver grafts using biliary tree and foetal hepatocytes.

A whole-organ regeneration approach, using a decellularised xenogeneic liver as a scaffold for the construction of a transplantable liver was recently reported. Deriving suitable scaffolds was the first step towards clinical application; however, effective recellularisation remains to be achieved. This report presents a strategy for the improvement of the recellularisation process, using novel cell-seeding technique and cell source. We evaluated recellularised liver grafts repopulated through the portal vein or the biliary duct with mice adult hepatocytes or E14.5 foetal hepatocytes. More than 80% of the cells seeded through the biliary tree entered the parenchyma beyond the ductule-lining matrix barrier and distributed throughout the liver lobule. In contrast, about 20% of the cells seeded through the portal tree entered the parenchyma. The gene expression levels of foetal hepatocyte albumin, glucose 6-phosphatase, transferrin, cytokeratin 19, and gamma-glutamyl transpeptidase were increased in three-dimensional cultures in the native liver-derived scaffolds, and the activation of liver detoxification enzymes and formation of biliary duct-like structures were supported. The metabolic functions of liver grafts recellularised with different cell types were similar. These results suggest that biliary tree cell-seeding approach is promising, and that liver progenitor cells represent a good cell source candidate.

SOX9 is a novel cancer stem cell marker surrogated by osteopontin in human hepatocellular carcinoma.

The current lack of cancer stem cell (CSC) markers that are easily evaluated by blood samples prevents the establishment of new therapeutic strategies in hepatocellular carcinoma (HCC). Herein, we examined whether sex determining region Y-box 9 (SOX9) represents a new CSC marker, and whether osteopontin (OPN) can be used as a surrogate marker of SOX9 in HCC. In HCC cell lines transfected with a SOX9 promoter-driven enhanced green fluorescence protein gene, FACS-isolated SOX9(+) cells were capable of self-renewal and differentiation into SOX9(-) cells, and displayed high proliferation capacity in vitro. Xenotransplantation experiments revealed that SOX9(+) cells reproduced, differentiated into SOX9(-) cells, and generated tumors at a high frequency in vivo. Moreover, SOX9(+) cells were found to be involved in epithelial-mesenchymal transition (EMT) and activation of TGFb/Smad signaling. Gain/loss of function experiments showed that SOX9 regulates Wnt/beta-catenin signaling, including cyclin D1 and OPN. Immunohistochemistry of 166 HCC surgical specimens and serum OPN measurements showed that compared to SOX9(-) patients, SOX9(+) patients had significantly poorer recurrence-free survival, stronger venous invasion, and higher serum OPN levels. In conclusion, SOX9 is a novel HCC-CSC marker regulating the Wnt/beta-catenin pathway and its downstream target, OPN. OPN is a useful surrogate marker of SOX9 in HCC.

Mucosal necrosis of the small intestine in myopathy, encephalopathy, lactic acidosis, and stroke-like episodes syndrome.

This report presents a case of massive mucosal necrosis of the small intestine in a patient with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), which particularly affects the brain, nervous system and muscles. A 45-year-old Japanese female, with an established diagnosis of MELAS, presented with vomiting. Computed tomography showed portomesenteric venous gas and pneumatosis intestinalis. She underwent a resection of the small intestine. A microscopic study showed necrosis of the mucosa and vacuolar degeneration of smooth muscle cells in the arterial wall. Immunohistochemistry showed anti-mitochondrial antibody to be highly expressed in the crypts adjacent the necrotic mucosa. The microscopic and immunohistochemical findings suggested the presence of a large number of abnormal mitochondria in MELAS to be closely linked to mucosal necrosis of the small intestine.

Adenocarcinoma in an intrapancreatic accessory spleen: report of a case.

We report a case of adenocarcinoma in an intrapancreatic accessory spleen (IPAS). A 78-year-old woman presented with abdominal discomfort, and investigations revealed an elevated serum carbohydrate antigen 19-9 level, to 161.8 U/ml (normal, <37 U/ml). Ultrasonography showed a heterogeneous echogenic tumor with a vascular hilum. Computed tomography showed a heterogeneously enhanced tumor, 8 cm in diameter, adjacent to the pancreatic body, accompanying a feeding artery arising from the splenic artery, and a drainage vein flowing into the splenic vein. We performed a distal pancreaticosplenectomy. The tumor was surrounded by a fibrous capsule and was in contact with the pancreatic body. Histological examinations revealed invasive growth of adenocarcinoma in a structure identical to the spleen. The results of both radiological and histological examinations suggested that the tumor originated from an intrapancreatic accessory spleen. Extensive examinations revealed no other malignancy, based on which we concluded that the adenocarcinoma was primary. Surgical intervention is strongly recommended when a malignancy in an IPAS cannot be ruled out.

Xanthogranulomatous pancreatitis mimicking a malignant cystic tumor of the pancreas: report of a case.

Xanthogranulomatous pancreatitis (XGP) is a rare inflammatory disease of the pancreas. A correct diagnosis is usually made only after pathological examination. A 76-year-old man was referred to our hospital for investigation of erythroderma, muscle weakness, and weight loss. We suspected dermatomyositis as a paraneoplastic phenomenon and investigated accordingly. Computed tomography showed a cystic lesion encapsulated by a thick wall in the pancreatic body. On magnetic resonance imaging, the lesion had low intensity on the T1-weighted images and heterogeneously high intensity on the T2-weighted images. (18)F-Fluorodeoxyglucose positron emission tomography showed abnormal uptake with a maximum standardized uptake value of 9.1. Based on these findings, we made a preoperative diagnosis of intraductal papillary-mucinous carcinoma and performed a distal pancreatectomy. Macroscopically, the cyst was surrounded by a yellow-tan mass with an unclear border, and was filled with hemorrhagic and necrotic tissue. Microscopically, the mass contained an aggregation of many foamy histiocytes, lymphocytes, and plasma cells. These microscopic findings were consistent with xanthogranulomatous inflammation, and the lesion was diagnosed as XGP. Although it is a rare benign pancreatic lesion, XGP should nevertheless be considered in the differential diagnosis of cystic lesions of the pancreas.