Hideaki Obara

Human-Scale Whole-Organ Bioengineering for Liver Transplantation: A Regenerative Medicine Approach

At this time, the only definitive treatment of hepatic failure is liver transplantation. However, transplantation has been limited by the severely limited supply of human donor livers. Alternatively, a regenerative medicine approach has been recently proposed in rodents that describe the production of three-dimensional whole-organ scaffolds for assembly of engineered complete organs. In the present study, we describe the decellularization of porcine livers to generate liver constructs at a scale that can be clinically relevant. Adult ischemic porcine livers were successfully decellularized using a customized perfusion protocol, the decellularization process preserved the ultrastructural extracellular matrix components, functional characteristics of the native microvascular and the bile drainage network of the liver, and growth factors necessary for angiogenesis and liver regeneration. Furthermore, isolated hepatocytes engrafted and reorganized in the porcine decellularized livers using a human-sized organ culture system. These results provide proof-of-principle for the generation of a human-sized, three-dimensional organ scaffold as a potential structure for human liver grafts reconstruction for transplantation to treat liver disease.

Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit

IntroductionSepsis is a serious medical condition that requires rapidly administered, appropriate antibiotic treatment. Conventional methods take three or more days for final pathogen identification and antimicrobial susceptibility testing. We organized a prospective observational multicenter study in three study sites to evaluate the diagnostic accuracy and potential clinical utility of the SeptiFast system, a multiplex pathogen detection system used in the clinical setting to support early diagnosis of bloodstream infections.MethodsA total of 212 patients, suspected of having systemic inflammatory response syndrome (SIRS) caused by bacterial or fungal infection, were enrolled in the study. From these patients, 407 blood samples were taken and blood culture analysis was performed to identify pathogens. Whole blood was also collected for DNA Detection Kit analysis immediately after its collection for blood culture. The results of the DNA Detection Kit, blood culture and other culture tests were compared. The chosen antimicrobial treatment in patients whose samples tested positive in the DNA Detection Kit and/or blood culture analysis was examined to evaluate the effect of concomitant antibiotic exposure on the results of these analyses.ResultsSeptiFast analysis gave a positive result for 55 samples, while 43 samples were positive in blood culture analysis. The DNA Detection Kit identified a pathogen in 11.3% (45/400) of the samples, compared to 8.0% (32/400) by blood culture analysis. Twenty-three pathogens were detected by SeptiFast only; conversely, this system missed five episodes of clinically significant bacteremia (Methicillin-resistant Staphylococcus aureus (MRSA), 2; Pseudomonas aeruginosa, 1; Klebsiella spp, 1; Enterococcus faecium, 1). The number of samples that tested positive was significantly increased by combining the result of the blood culture analysis with those of the DNA Detection Kit analysis (P = 0.01). Among antibiotic pre-treated patients (prevalence, 72%), SeptiFast analysis detected more bacteria/fungi, and was less influenced by antibiotic exposure, compared with blood culture analysis (P = 0.02).ConclusionsThis rapid multiplex pathogen detection system complemented traditional culture-based methods and offered some added diagnostic value for the timely detection of causative pathogens, particularly in antibiotic pre-treated patients. Adequately designed intervention studies are needed to prove its clinical effectiveness in improving appropriate antibiotic selection and patient outcomes.

Overexpression of Truncated IκBα Induces TNF-α–Dependent Apoptosis in Human Vascular Smooth Muscle Cells

Dysregulation of apoptosis is one of the likely underlying mechanisms of neointimal thickening, a disorder in which proinflammatory cytokines may influence the function of vascular smooth muscle cells (VSMCs) and contribute to atherogenesis. One of these cytokines, tumor necrosis factor-alpha (TNF-alpha), induces 2 possibly conflicting pathways, 1 leading to the activation of nuclear factor-kappaB (NF-kappaB) and the other leading to caspase-mediated apoptosis. We investigated whether specific inhibition of NF-kappaB affects TNF-alpha-dependent apoptosis in human VSMCs. To inhibit NF-kappaB activation specifically, we constructed a recombinant adenovirus vector expressing a truncated form of the inhibitor protein IkappaBalpha (AdexIkappaBDeltaN) that lacks the phosphorylation sites essential for activation of NF-kappaB. The IkappaBDeltaN was overexpressed by adenoviral infection and was resistant to stimulus-dependent degradation. Electromobility gel shift and luciferase assays demonstrated that overexpression of IkappaBDeltaN inhibited NF-kappaB activation induced by TNF-alpha or interleukin-1beta (IL-1beta). In cells overexpressing IkappaBDeltaN, TNF-alpha dramatically induced apoptosis, whereas IL-1beta had no effect. The induction was suppressed by treatment with a selective inhibitor of the caspase-3 family, Z-DEVD-fmk, and the overexpression of IkappaBDeltaN induced TNF-alpha-mediated caspase-3 and caspase-2 activity. These results indicate that overexpression of IkappaBDeltaN induces TNF-alpha-dependent apoptosis by efficient and specific suppression of NF-kappaB and upregulation of caspase-3 and caspase-2 activity in human VSMCs. Our findings suggest that adenovirus-mediated IkappaBDeltaN gene transfer may be useful in the treatment of disorders associated with inflammatory conditions, such as the response to vascular injury and atherosclerosis.

The influence of donor age on liver regeneration and hepatic progenitor cell populations.

BACKGROUND Recent reports suggest that donor age might have a major impact on recipient outcome in adult living donor liver transplantation (LDLT), but the reasons underlying this effect remain unclear. The aims of this study were to compare liver regeneration between young and aged living donors and to evaluate the number of Thy-1+ cells, which have been reported to be human hepatic progenitor cells. METHODS LDLT donors were divided into 2 groups (Group O, donor age ≥ 50 years, n = 6 and Group Y, donor age ≤ 30 years, n = 9). The remnant liver regeneration rates were calculated on the basis of computed tomography volumetry on postoperative days 7 and 30. Liver tissue samples were obtained from donors undergoing routine liver biopsy or patients undergoing partial hepatectomy for metastatic liver tumors. Thy-1+ cells were isolated and counted using immunomagnetic activated cell sorting (MACS) technique. RESULTS Donor liver regeneration rates were significantly higher in young donors compared to old donors (P = .042) on postoperative day 7. Regeneration rates were significantly higher after right lobe resection compared to rates after left lobe resection. The MACS findings showed that the number of Thy-1+ cells in the human liver consistently tended to decline with age. CONCLUSION Our study revealed that liver regeneration is impaired with age after donor hepatectomy, especially after right lobe resection. The declining hepatic progenitor cell population might be one of the reasons for impaired liver regeneration in aged donors.

Mesenchymal stem cells support hepatocyte function in engineered liver grafts

Recent studies suggest that organ decellularization is a promising approach to facilitate the clinical application of regenerative therapy by providing a platform for organ engineering. This unique strategy uses native matrices to act as a reservoir for the functional cells which may show therapeutic potential when implanted into the body. Appropriate cell sources for artificial livers have been debated for some time. The desired cell type in artificial livers is primary hepatocytes, but in addition, other supportive cells may facilitate this stem cell technology. In this context, the use of mesenchymal stem cells (MSC) is an option meeting the criteria for therapeutic organ engineering. Ideally, supportive cells are required to (1) reduce the hepatic cell mass needed in an engineered liver by enhancing hepatocyte function, (2) modulate hepatic regeneration in a paracrine fashion or by direct contact, and (3) enhance the preservability of parenchymal cells during storage. Here, we describe enhanced hepatic function achieved using a strategy of sequential infusion of cells and illustrate the advantages of co-cultivating bone marrow-derived MSCs with primary hepatocytes in the engineered whole-liver scaffold. These co-recellularized liver scaffolds colonized by MSCs and hepatocytes were transplanted into live animals. After blood flow was established, we show that expression of adhesion molecules and proangiogenic factors was upregulated in the graft.

Protective effect of high-mobility group box 1 blockade on acute liver failure in rats

High-mobility group box 1 (HMGB1) is a monocyte-derived inflammatory mediator that is released in some conditions including shock, tissue injury, and endotoxin-induced lethality. In this study, we determined the plasma and hepatic tissue levels of HMGB1 in a drug-induced rat acute liver failure (ALF) model and investigated the effect of HMGB1 blockade on ALF. Adult male Sprague-Dawley rats, weighing 250 to 300 g, were used for this study. d-galactosamine was injected into the penile vein to induce ALF. To determine HMGB1 levels, plasma and hepatic tissue samples were serially collected after the d-galactosamine injection. To test the effect of HMGB1 blockade, anti-HMGB1 polyclonal antibodies or control antibodies were injected into the penile vein right after injection of d-galactosamine. Levels of HMGB1 were increased in plasma and decreased in hepatic tissue after induction of ALF. Immunohistochemical examination for HMGB1 showed that liver from animals with ALF had little staining, whereas normal liver had strong staining in the nuclei. Injection of anti-HMGB1 antibodies resulted in significant suppression of plasma HMGB1 and hepatic enzymes, marked suppression of plasma inflammatory cytokines, marked improvement of histological findings, and significant improvement of survival. The decrease of hepatic HMGB1 was also significantly suppressed in the group injected with anti-HMGB1 antibodies. The present study suggests that in ALF, the liver may release HMGB1 into the plasma, and that neutralizing the released HMGB1 has a protective effect against injury.ABBREVIATIONS-ALF-acute liver failure; HMGB1-high-mobility group box 1; ELISA-enzyme-linked immunosorbent assay; anti-HMGB1 Ab-anti-HMGB1 polyclonal antibodies; control Ab-control IgY antibodies; AST-aspartate aminotransferase; ALT-alanine aminotransferase; LDH-lactic dehydrogenase; IL-interleukin; TNF-tumor necrosis factor

Surgical strategy for abdominal aortic aneurysm with concurrent symptomatic malignancy.

In an attempt to improve the guidelines for concurrent management of concomitant abdominal aortic aneurysm (AAA) and symptomatic malignancy, a retrospective study was undertaken. A total of 186 AAA repairs were performed electively, and 25 patients (13.4%) had concurrent symptomatic malignancy from April 1986 to March 1997. Fourteen patients underwent a one-stage operation, including five abdominoperineal rectal resections, four subtotal gastrectomies, three total gastrectomies, and two right hemicolectomies. Eleven others underwent a two-stage operation, including four with total gastrectomy and left hemicolectomy followed by AAA repair, as well as two with right hemicolectomy and one with left hemicolectomy prior to AAA repair. There were no operative deaths or severe postoperative complications. Of the 25 patients, 22 (88.0%) are still alive during follow-up ranging from 8 months to 11 years. Our surgical approach to both lesions is as follows: (1) Using the transperitoneal approach alone, subtotal gastrectomy and abdominoperineal rectal resection can be safely done simultaneously. (2) Although total gastrectomy can also be performed concurrently, the approach used for each lesion is separate. (3) Colorectal resection is generally done separately. However, a one-stage operation can be performed using the thromboexclusion procedure for AAA repair in patients with right-sided colonic cancer or a temporary transverse colostomy for left-sided colorectal cancer.

Evolution of Computed Tomographic Characteristics of Spontaneous Isolated Superior Mesenteric Artery Dissection During Conservative Management

BACKGROUND Spontaneous isolated superior mesenteric artery (SMA) dissection is a rare condition, and its clinical and angiographic courses are poorly defined. We aimed to monitor the morphological characteristics of spontaneous isolated SMA dissection using computed tomography (CT) over 2 years of follow-up, including the recovery process via vascular remodeling, and identify the factors that affect vascular remodeling using univariate analysis. METHODSANDRESULTS We retrospectively reviewed the medical records and morphological findings of 59 consecutive patients with spontaneous isolated SMA dissection between October 2007 and July 2014, which included 36 symptomatic and 23 asymptomatic patients. Surgical intervention with open laparotomy was required in 3 patients during the follow-up period; 41 patients who received conservative treatment were followed up over 2 years with regular CT. Complete remodeling was achieved in 16 of 25 symptomatic patients who were treated conservatively (64.0%). A patent false lumen and aneurysmal formation on an initial CT scan were identified as negative factors that affected remodeling in patients with spontaneous isolated SMA dissection. CONCLUSIONS Conservative management of spontaneous isolated SMA dissection is associated with a good prognosis, both clinically and morphologically. Surgical intervention is only required in patients with severe intestinal ischemia or rapid aneurysmal enlargement. (Circ J 2016; 80: 1452-1459).

Role of high mobility group box chromosomal protein 1 in ischemia-reperfusion injury in the rat small intestine

BACKGROUND High mobility group box chromosomal protein 1 (HMGB1) has recently been shown to be an important late mediator of endotoxic shock and sepsis. The purpose of the present study was to investigate the role of HMGB1 in response to ischemia-reperfusion injury. METHODS Ischemia-reperfusion injury was induced in male Wistar rats by clamping the superior mesenteric artery for 60 min. Using this model, the serum concentrations and localization of HMGB1 were investigated. The histologic findings from reperfused intestines and the survival rates were compared between the anti-HMGB1 antibody treatment groups (group A treated with 6.0 mg/kg antibody and group B with 0.6 mg/kg antibody) and the control antibody treatment group (control group). RESULTS Serum HMGB1 concentrations increased early after reperfusion and peaked at 3 h. Immunohistochemistry for HMGB1 revealed a high degree of positive staining in the epithelial cells of the damaged villi. Anti-HMGB1 antibody treatment significantly reduced this damage (P < 0.05) and improved the 48-h survival rate (90% in group A versus 50% in the controls; P < 0.05). CONCLUSIONS These results suggest that HMGB1 plays a key role in small intestinal ischemia-reperfusion injury.

Celiomesenteric anomaly with concurrent aneurysm

We describe a rare case of a celiomesenteric anomaly with concurrent aneurysm. The patient, a 53-year-old man, had no abdominal pain or discomfort. The presence of a celiac artery aneurysm was suspected on the basis of the results of abdominal computerized tomographic scanning and echo ultrasound scanning performed because of proteinuria. Intra-arterial digital subtraction angiographic results showed the anomaly and aneurysm. Because of the risk of rupture of the aneurysm, the lesion was repaired surgically, with the placement of an interpositional prosthetic graft. We found no previous reports of celiomesenteric anomaly with concurrent aneurysm repaired with prosthetic graft.