Intestinal epithelial culture under an air-liquid interface: a tool for studying human and mouse esophagi.

This study investigated whether an intestinal epithelial culture method can be applied to mouse and human esophageal cultures. The esophagi harvested from 1-day-old mice and adult humans were maintained in collagen gels. A commercially available culture medium for human embryonic stem cells was used for the human esophageal culture. We discovered that the intestinal epithelial culture method can be successfully applied to both mouse and human esophageal cultures. The long-term cultured esophageal organoids were rod-like luminal structures lined with myofibroblasts. We discovered that regeneration of the esophageal mucosal surface can be almost completely achieved in vitro, and the advantage of this method is that organoid cultures may be generated using host-derived fibroblasts as a niche. This method is a promising tool for mouse and human research in intestinal biology, carcinogenesis, and regenerative medicine.

Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer.

BACKGROUND: Functional microRNAs (miRNAs) in exosomes have been recognised as potential stable biomarkers in cancers. The aim of this study is to identify specific miRNAs in exosome as serum biomarkers for the early detection of recurrence in human colorectal cancer (CRC). METHODS: Serum samples were sequentially obtained from six patients with and without recurrent CRC. The miRNAs were purified from exosomes, and miRNA microarray analysis was performed. The miRNA expression profiles and copy number aberrations were explored using microarray and array CGH analyses in 124 CRC tissues. Then, we validated exosomal miRNAs in 2 serum sample sets (90 and 209 CRC patients) by quantitative real-time RT-PCR. RESULTS: Exosomal miR-17-92a cluster expression level in serum was correlated with the recurrence of CRC. Exosomal miR-19a expression levels in serum were significantly increased in patients with CRC as compared with healthy individuals with gene amplification. The CRC patients with high exosomal miR-19a expression showed poorer prognoses than the low expression group (P<0.001). CONCLUSIONS: Abundant expression of exosomal miR-19a in serum was identified as a prognostic biomarker for recurrence in CRC patients.

Circulating tumour cell-derived plastin3 is a novel marker for predicting long-term prognosis in patients with breast cancer.

BACKGROUND: Identification of promising biomarkers that predict the prognosis of patients with breast cancer is needed. In this study, we hypothesised that the expression of the epithelial-mesenchymal transition-related biomarker plastin3 (PLS3) in peripheral blood could be a prognostic factor in breast cancer. METHODS: We examined PLS3 expression in breast cancer cell lines with epithelial and mesenchymal traits and in circulating tumour cells (CTCs) obtained from the peripheral blood of breast cancer patients. We investigated PLS3 expression in the peripheral blood of 594 patients with breast cancer to evaluate the clinical significance of PLS3 expression. RESULTS: Robust PLS3 expression was observed in different breast cancer cell lines (Hs578t, MCF-7, MDA-MB-468, and MDA-MB-231) as well as in a bone marrow derived cancer cell line (BC-M1). In both the training (n=298) and validation (n=296) sets, PLS3 expression was observed in CTCs of patients with breast cancer. PLS3-positive patients showed significantly poorer overall and disease-free survival than PLS3-negative patients (P=0.0001 and 0.003, respectively). Subset analysis revealed that this prognostic biomarker was relevant in patients with stage I-III cancer, particularly in patients with luminal-type and triple-negative-type tumours. CONCLUSIONS: These data demonstrated that PLS3 was expressed in CTCs undergoing the epithelial-mesenchymal transition in patients with breast cancer. Furthermore, PLS3 may be an excellent biomarker for identifying groups at risk of recurrence or with a poor prognosis.

The AURKA/TPX2 axis drives colon tumorigenesis cooperatively with MYC.

BACKGROUND: The MYC oncogene has long been established as a central driver in many types of human cancers including colorectal cancer. However, the realization of MYC-targeting therapies remains elusive; as a result, synthetic lethal therapeutic approaches are alternatively being explored. A synthetic lethal therapeutic approach aims to kill MYC-driven tumors by targeting a certain co-regulator on the MYC pathway. PATIENTS AND METHODS: We analyzed copy number and expression profiles from 130 colorectal cancer tumors together with publicly available datasets to identify co-regulators on the MYC pathway. Candidates were functionally tested by in vitro assays using colorectal cancer and normal fibroblast cell lines. Additionally, survival analyses were carried out on another 159 colorectal cancer patients and public datasets. RESULTS: Our in silico screening identified two MYC co-regulator candidates, AURKA and TPX2, which are interacting mitotic regulators located on chromosome 20q. We found the two candidates showed frequent co-amplification with the MYC locus while expression levels of MYC and the two genes were positively correlated with those of MYC downstream target genes across multiple cancer types. In vitro, the aberrant expression of MYC, AURKA and TPX2 resulted in more aggressive anchorage-independent growth in normal fibroblast cells. Furthermore, knockdown of AURKA or TPX2, or treatment with an AURKA-specific inhibitor effectively suppressed the proliferation of MYC-expressing colorectal cancer cells. Additionally, combined high expression of MYC, AURKA and TPX2 proved to be a poor prognostic indicator of colorectal cancer patient survival. CONCLUSIONS: Through bioinformatic analyses and experiments, we proposed TPX2 and AURKA as novel co-regulators on the MYC pathway. Inhibiting the AURKA/TPX2 axis would be a novel synthetic lethal therapeutic approach for MYC-driven cancers.

Identification of a bona fide microRNA biomarker in serum exosomes that predicts hepatocellular carcinoma recurrence after liver transplantation.

BACKGROUND: Predictive biomarkers for the recurrence of hepatocellular carcinoma (HCC) have great benefit in the selection of treatment options, including liver transplantation (LT), for HCC. The purpose of this study was to identify specific microRNAs (miRs) in exosomes from the serum of patients with recurrent HCC and to validate these molecules as novel biomarkers for HCC recurrence. METHODS: We employed microarray-based expression profiling of miRs derived from exosomes in the serum of HCC patients to identify a biomarker that distinguishes between patients with and without HCC recurrence after LT. This was followed by the validation in a separate cohort of 59 HCC patients who underwent living related LT. The functions and potential gene targets of the recurrence-specific miRs were analysed using a database, clinical samples and HCC cell lines. RESULTS: We found that miR-718 showed significantly different expression in the serum exosomes of HCC cases with recurrence after LT compared with those without recurrence. Decreased expression of miR-718 was associated with HCC tumour aggressiveness in the validated cohort series. We identified HOXB8 as a potential target gene of miR-718, and its upregulation was associated with poor prognosis. CONCLUSION: Circulating miRs in serum exosomes have potential as novel biomarkers for predicting HCC recurrence.

Amplification of PVT-1 is involved in poor prognosis via apoptosis inhibition in colorectal cancers.

BACKGROUND: We previously conducted gene expression microarray analyses to identify novel indicators for colorectal cancer (CRC) metastasis and prognosis from which we identified PVT-1 as a candidate gene. PVT-1, which encodes a long noncoding RNA, mapped to chromosome 8q24 whose copy-number amplification is one of the most frequent events in a wide variety of malignant diseases. However, PVT-1 molecular mechanism of action remains unclear. METHODS: We conducted cell proliferation and invasion assays using colorectal cancer cell lines transfected with PVT-1siRNA or negative control siRNA. Gene expression microarray analyses on these cell lines were also carried out to investigate the molecular function of PVT-1. Further, we investigated the impact of PVT-1 expression on the prognosis of 164 colorectal cancer patients by qRT-PCR. RESULTS: CRC cells transfected with PVT-1 siRNA exhibited significant loss of their proliferation and invasion capabilities. In these cells, the TGF-ß signalling pathway and apoptotic signals were significantly activated. In addition, univariate and multivariate analysis revealed that PVT-1 expression level was an independent risk factor for overall survival of colorectal cancer patients. CONCLUSION: PVT-1, which maps to 8q24, generates antiapoptotic activity in CRC, and abnormal expression of PVT-1 was a prognostic indicator for CRC patients.

PICT1 regulates TP53 via RPL11 and is involved in gastric cancer progression.

BACKGROUND: The TP53 pathway is frequently inactivated in human cancers. PICT1 (also known as GLTSCR2) is a novel regulator of the MDM2-TP53 pathway via its interaction with the ribosomal protein RPL11 in the nucleolus. However, the clinical significance of PICT1 in gastric cancer remains unknown. METHODS: To evaluate PICT1 function, we used shRNA to inhibit PICT1 expression in gastric cancer cells that expressed wild-type TP53. PICT1 expression and TP53 mutation status were quantified in 110 cases of primary gastric cancer to explore the impact of PICT1 expression levels on gastric cancer. RESULTS: Deficiency of PICT1 significantly impaired cell proliferation and colony formation via TP53-mediated cell cycle arrest. Following induction of PICT1 deficiency, RPL11 translocated out of the nucleolus. Of the 110 gastric cancer samples tested, 70 (63.6%) and 40 (36.4%) tumours expressed wild-type and mutant TP53, respectively. In gastric cancer patients with wild-type TP53 tumours, patients with relatively low PICT1 expression levels had a better prognosis compared with high expression level patients (P=0.046). CONCLUSION: The findings suggest that PICT1 has a crucial role in gastric cancer progression by regulating the MDM2-TP53 pathway through RPL11. Clinically, PICT1 expression is a novel prognostic parameter in gastric cancer patients with wild-type TP53 tumours.

Paired related homoeobox 1, a new EMT inducer, is involved in metastasis and poor prognosis in colorectal cancer.

BACKGROUND: Paired related homoeobox 1 (PRRX1) has been identified as a new epithelial-mesenchymal transition (EMT) inducer in breast cancer. However, the function of PRRX1 in colorectal cancer (CRC) has not been elucidated. METHODS: We utilised ectopic PRRX1-expressing cell lines to analyse the function of PRRX1 in CRC. The clinical significance of PRRX1 was also examined on three independent CRC case sets. RESULTS: PRRX1 induced EMT and the stem-like phenotype in CRC cells. In contrast to studies of breast cancer, abundant expression of PRRX1 was significantly associated with metastasis and poor prognosis in CRC. CONCLUSION: PRRX1 is an indicator of metastasis and poor prognosis in CRC cases. Further investigation is required to uncover the signalling network regulating PRRX1.

End-stage liver cirrhosis with severe autoimmune hemolytic anemia, treated by blood type-incompatible living donor liver transplantation: a case report.

We present a case of successful living donor liver transplantation (LDLT) for liver cirrhosis caused by hepatitis B virus with severe autoimmune hemolytic anemia (AIHA) using an ABO-incompatible (ABOi) graft. The patient was a 47-year-old woman who had a history of ruptured esophageal varices, accumulation of intractable ascites, frequent hepatic encephalopathy and severe anemia, with a hemoglobin value of approximately 3 g/dL due to AIHA. We treated the patient by LDLT using an ABOi liver graft. The treatment strategy included anti-CD20 antibody, plasma exchange and transfusion before LDLT. The patient's anemia improved after surgery; she required only 2 units of irradiated red blood cell concentrates-leukocytes reduced. The patient was discharged from the hospital on postoperative day 35. Two years after surgery, the patient still shows normal hepatic and hematological findings. The immunomodulation protocol for ABOi LDLT was effective not only to avoid humoral reactions associated with ABOi LDLT, but also those associated with AIHA.

Rapid normalization of portopulmonary hypertension after living donor liver transplantation.

Portopulmonary hypertension (PPHTN) is a relatively rare complication of end-stage liver disease, and a serious problem in the context of liver transplantation. Herein we have reported a case of decompensated liver cirrhosis with PPHTN, which rapidly resolved after adult-to-adult living donor liver transplantation (LDLT). A 54-year-old man was referred to our hospital with end-stage liver cirrhosis owing to chronic hepatitis C. Preoperative mean pulmonary artery pressure (mPAP), as assessed by right heart catheterization, was 38 mm Hg. Continuous infusion of epoprostenol decreased the mPAP to 24 mm Hg over 44 days. He underwent LDLT using a right hepatic lobe graft donated by his son. The postoperative course was uneventful, epoprostenol was weaned by postoperative day (POD) 21, and the mPAP normalized to 21 mm Hg on POD 28. The patient was discharged on POD 31 without any vasodilators. Our case revealed that liver transplantation can rapidly resolve PPTHN.

Sequential pancreaticoduodenectomy after living donor liver transplantation for cholangiocarcinoma.

Liver transplantation (LT) for patients with primary sclerosing cholangitis (PSC) is often contraindicated due to concomitant occurrence of cholangiocarcinoma (CC). Cases of simultaneous pancreaticoduodenectomy (PD) with LT have been sporadically reported; however, the applicability of such an invasive procedure to patients with CC has not been validated. We report here a case of sequential PD performed 44 days after a successful living donor liver transplantation (LDLT) using a left lobe graft. Although a clear pancreatic juice leakage through the drain persisted for days after surgery, the patient recovered from the complication and was discharged 32 days after the procedure. Currently, 1 year after LDLT, the patient is doing well with no evidence of recurrence. In conclusion, a sequential PD following LDLT is a safe and feasible option to treat CC complicating PSC. Long-term follow-up and accumulation of cases are necessary to evaluate the effectiveness of this procedure for this complicated disease.

Characterization of adipose tissue-derived cells isolated with the Celution system.

BACKGROUND: The therapeutic potential of using stem cells is tremendous. Mesenchymal stromal cells (MSC) have now been isolated in various tissues including bone marrow (BM), muscle, skin and adipose tissue. Among them, adipose tissue could be one of the most suitable cell sources for cell therapy, because of its easy accessibility, minimal morbidity and abundance of stem cells. The large numbers of stem cells in adipose tissue means that clinically relevant stem cell numbers could be extracted from the tissue, potentially eliminating the need for in vitro expansion. To utilize these characteristics of adipose tissue fully, Cytori Therapeutics Inc. has developed a closed system called Celution to isolate and concentrate stem cells and regenerative cells automatically from adipose tissue. METHODS: Adipose tissue-derived cells were isolated using the Celution system. The output from the Celution was characterized using multicolor FACS analysis with CD31, CD34, CD45, CD90, CD105 and CD146. The multidifferentiation potential of the cells was analyzed using adipogenic and osteogenic media. RESULTS: Our results showed that cells from the Celution are composed of heterogeneous cell populations including adipose-derived stem cells (ASC) (CD31- CD34+ CD45- CD90+ CD105- CD146-), endothelial (progenitor) cells (CD31+ CD34+ CD45- CD90+ CD105- CD146+) and vascular smooth muscle cells (CD31- CD34+ CD45- CD90+ CD105- CD146+). We also confirmed the output contains cells able to differentiate into adipogenic and osteogenic phenotypes. Our results show that cells isolated with the Celution and manually are equivalent. DISCUSSION: Cells from adipose tissue can be processed by Celution within the time frame of a single surgical procedure. This system could provide a 'real-time' treatment setting that is cost-effective and safe.

Reduced tau expression in gastric cancer can identify candidates for successful Paclitaxel treatment.

A recent study disclosed that breast cancer cases with low 'tau' expression can predict susceptibility to Paclitaxel administration. In the current study, the clinical significance of tau expression in gastric cancer cases was established by identifying candidates with Paclitaxel administration. Tissue specimens from 20 cases of in-operable or noncuratively resected gastric cancer were examined. Subsequent to the administration of 80 mg m(-2) of Paclitaxel in six 3-h intravenous infusions, the clinical effectiveness of Paclitaxel was evaluated by the size of metastatic lesions with computed tomography. The status of the tau expression was determined by immunohistochemistry. Based on a previously reported classification scheme, six were classified as tau-negative expression (0, 1+) cases and 14 were classified as tau-positive expression (2+, 3+) cases. All six (100%) cases of tau-negative expression showed a favourable response (partial response or minor response) to Paclitaxel administration. However, 12 (86%) of the 14 cases of tau-positive expression showed progressive disease (n = 11) or no change (n = 1) after Paclitaxel administration. The serum carcinoembryonic antigen values of the six cases of tau-negative expression were markedly decreased in comparison to the 14 tau-positive cases. These data indicate that tau-negative expression can be used to select gastric cancer patients, which will favourably respond to Paclitaxel treatment.

Angiopoietin switching regulates angiogenesis and progression of human hepatocellular carcinoma.

AIM: Angiopoietin 1 (Ang-1) and its antagonist, angiopoietin 2 (Ang-2), are novel ligands that regulate the Tie2 receptor. The Ang-2 gene is upregulated in the hypervascular type of human hepatocellular carcinoma (HCC). To gain a better understanding of the role of the Ang-Tie2 system in HCC the expression of these genes was investigated in a series of human HCCs. METHODS: The expression of the angiopoietin and Tie2 proteins was investigated in nine normal liver tissues and 52 surgically resected HCCs. In addition, the effects of hypoxic stimuli on Ang-1, Ang-2, vascular endothelial growth factor (VEGF), and erythropoietin (EPO) expression was investigated in Hep3B cells. RESULTS: Ang-1, rather than Ang-2, was more frequently expressed in the normal liver. Ang-1 was expressed in 68% of HCCs, whereas Ang-2 was expressed in 81%, and was significantly higher in poorly differentiated HCCs characterised by high vascularity (p = 0.02), and in tumours with a peliotic change (p = 0.02). Strong expression of Tie2 was seen in tumour vessels in accordance with Ang-2 expression. In Hep3B cells, hypoxic stimuli upregulated VEGF and EPO, but not Ang-1 or Ang-2. CONCLUSIONS: These data support the evidence that the reversal of Ang-1 and Ang-2 expression plays an important role in the angiogenic and dedifferentiation processes in HCC. The hypoxic stimuli were not responsible for Ang-2 upregulation, unlike that of VEGF, in human HCC cells.

Recombinant Sendai virus vectors for activated T lymphocytes.

T-lymphocyte-directed gene therapy has potential as a treatment of subjects with immunological disorders. One current limitation of this therapeutic strategy is low gene transfer efficiency, even when complex procedures are used. We report herein that a recombinant Sendai virus vector (SeV) was able to overcome this issue. Using jellyfish enhanced green fluorescent protein gene (EGFP), we found that SeV was able to transduce and express a foreign gene specifically and efficiently in activated murine and human T cells, but not in naive T cells, without centrifugation or reagents including polybrene and protamine sulfate; the present findings were in clear contrast to those demonstrated with the use of retroviruses. The transduction was selective in antigen-activated T cells, while antigen-irrelevant T cells were not transduced, even under bystander activation from specific T-cell responses by antigens ex vivo. Receptor saturation studies suggested a possible mechanism of activated T-cell-specific gene transfer, ie, SeV might attach to naive T cells but might be unable to enter their cytoplasm. We therefore propose that the SeV vector system may prove to be a potentially important alternative in the area of T-cell-directed gene therapy used in the clinical setting.

Efficacy of a polyurethane foam/spheroid artificial liver by using human hepatoblastoma cell line (Hep G2).

We invesigated the availability of human hepatoblastoma cell line (Hep G2), compared with human primary hepatocytes (HH) and porcine primary hepatocytes (PH), as a cell source for the hybrid artificial liver support system (HALSS) by using polyurethane foam (PUF). All three kinds of hepatocytes spontaneously formed spherical multicellular aggregates (spheroids) of 100-200 microm diameter in the pores of PUF within 3 days of culture. In a PUF stationary culture, Hep G2 spheroids recovered the ammonia removal activity that was lost in monolayer culture, although the removal for each unit cell number was about one tenth that of HH spheroids and about one eighth of PH spheroids. The synthesis activities of albumin and fibrinogen of each unit cell number of Hep G2 were also upregulated by PUF spheroid culture, and were about twice as high as in monolayer culture. The albumin secretion activity of Hep G2 spheroids was almost the same as that of PH spheroids. HH scarcely secreted these proteins in this experiment, probably because they were cultured in a serum-free medium. In the PUF module in a circulation culture, HH had high ammonia removal and low synthesis activities similar to stationary culture. Hep G2 proliferated to a high cell density, such as about 4.8 x 10(7) cells/cm3-module at 10 days of culture. Although Hep G2 spheroids had low ammonia removal activity in each cell, the removal rate in the PUF module was almost the same as for PH at 7 days of culture because of the high cell density culture by cell proliferation. The albumin secretion rate by Hep G2 in the PUF module also increased with cell proliferation and was about 10 times higher than the initial for the rate for PH at 7 days of culture. These results suggest that Hep G2 is a potential cell source PUF-HALSS.

Airway-directed gene transfer of interleukin-10 using recombinant Sendai virus effectively prevents post-transplant fibrous airway obliteration in mice.

Bronchiolitis obliterans (BO) after lung transplantation prevents a satisfactory prognosis, and recent studies suggested that interleukin-10 (IL-10) gene transfer to distant organs could inhibit BO in rodent models. Although delivery of the therapeutic gene to a local airway would be favored to minimize systemic effects, current limitations include lower gene transfer efficiency to airway epithelium. As recombinant Sendai virus (SeV) can produce dramatically efficient gene transfer to airway epithelium, we determined if SeV-mediated IL-10 gene transfer to the local airway would inhibit bronchial fibrous obliteration in murine tracheal allografts. Administration of cyclosporine A (CsA) significantly promoted not only recovery of the injured airway epithelium but also SeV-mediated IL-10 expression (CsA- versus CsA+ =228+/-78 versus 3627+/-1372 pg/graft with 5 x 10(7) pfu), thereby suggesting the requirement of epithelia for efficient gene transfer. Even at the highest expression, no significant leakage of IL-10 was evident in the systemic circulation, and the induction of interferon-gamma was completely diminished on day 7 by IL-10 gene transfer. As a result, luminal loss was significantly prevented in allografts treated with SeV-IL-10 (luminal opening, all control groups: 0% respectively, and SeV-IL-10 5 x 10(7) pfu: 25.7+/-10.5%), an effect that was enhanced by short-term CsA treatment (SeV-IL-10 5 x 10(7) pfu with CsA: 63.7+/-12.7%). We propose that SeV is a useful vector that can target airway epithelium to prevent BO avoiding putative systemic effect.

Laparoscopy-assisted hepatectomy using the Endoclose: a case report.

Hemostasis of a resected stump of liver is extremely difficult in laparoscopic hepatectomy. Although Pringle's maneuver, which is a total clamping of the hepatoduodenal ligament, is a useful technique, it is often difficult in laparoscopic circumstances. Moreover, total inflow occlusion leads to postoperative liver damage. Therefore, the local bleeding method is ideal. The Endoclose, a device for port site closure, is formed from an outer sheath and an inner needle with a notch to load the suture. The Endoclose is loaded with a suture and passed through the liver. The suture is left under the liver, and the device is removed. Next, the suture carrier is passed through the liver at an appropriate distance, and the suture is regrasped by this suture carrier and brought out of the liver. Herein we report a case in which a new bleeding control method using Endoclose was introduced for laparoscopy-assisted hepatectomy.