Characterizations of a neutralizing antibody broadly reactive to multiple gluten peptide:HLA-DQ2.5 complexes in the context of celiac disease.

In human celiac disease (CeD) HLA-DQ2.5 presents gluten peptides to antigen-specific CD4+ T cells, thereby instigating immune activation and enteropathy. Targeting HLA-DQ2.5 with neutralizing antibody for treating CeD may be plausible, yet using pan-HLA-DQ antibody risks affecting systemic immunity, while targeting selected gluten peptide:HLA-DQ2.5 complex (pHLA-DQ2.5) may be insufficient. Here we generate a TCR-like, neutralizing antibody (DONQ52) that broadly recognizes more than twenty-five distinct gluten pHLA-DQ2.5 through rabbit immunization with multi-epitope gluten pHLA-DQ2.5 and multidimensional optimization. Structural analyses show that the proline-rich and glutamine-rich motif of gluten epitopes critical for pathogenesis is flexibly recognized by multiple tyrosine residues present in the antibody paratope, implicating the mechanisms for the broad reactivity. In HLA-DQ2.5 transgenic mice, DONQ52 demonstrates favorable pharmacokinetics with high subcutaneous bioavailability, and blocks immunity to gluten while not affecting systemic immunity. Our results thus provide a rationale for clinical testing of DONQ52 in CeD.

Cardiopulmonary arrest upon admission caused by pilsicainide hydrochloride intoxication: A case report.

A 22-year-old male presented to our hospital after receiving 2450 mg of pilsicainide hydrochloride. Subsequently, he experienced cardiac arrest, and percutaneous cardiopulmonary support was introduced to maintain his circulation. After 3 days of intensive care, he regained consciousness and was transferred to another hospital for treatment related to psychological problems.

Corrigendum: Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3-mediated therapeutics.

This corrects the article DOI: 10.1038/srep45839.

Two cases of cryptogenic life-threatening hemoptysis - identification and management of bleeding point.

Cases: Case 1: A 63-year-old woman was referred for coughing blood. Although cardiorespiratory dynamics were stabilized by artificial respiration under sedation, severely poor ventilation developed from asphyxia associated with massive respiratory tract hemorrhage. One-lung ventilation was temporarily secured by endotracheal tube insertion into the left main bronchus just prior to cardiopulmonary arrest.Case 2: A 72-year-old man was referred for massive hemoptysis after coughing, then intubated and placed on a respirator. During angiography, blood clots collected with bronchoscopy confirmed extravascular leakage into the right main bronchus. Outcomes: Both showed no hemoptysis recurrence after bronchial artery embolization and were discharged. Case 1 required intensive treatment for 6 days, including artificial respiratory management. Conclusion: Emergency one-lung ventilation was required for asphyxia in Case 1, and we had difficulties with bleeding point identification and hemostatic therapy. From that experience, we noted hemoptysis during angiography using bronchoscopy in Case 2, enabling prompt bronchial artery embolization.

Entire CD3ε, δ, and γ humanized mouse to evaluate human CD3-mediated therapeutics.

T cell-mediated immunotherapy is an attractive strategy for treatment in various disease areas. In this therapeutic approach, the CD3 complex is one of the key molecules to modulate T cell functions; however, in many cases, we cannot evaluate the drug candidates in animal experiments because the therapeutics, usually monoclonal antibodies specific to human CD3, cannot react to mouse endogenous Cd3. Although immunodeficient mice transfused with human hematopoietic stem or precursor cells, known as humanized mice, are available for these studies, mice humanized in this manner are not completely immune competent. In this study we have succeeded in establishing a novel mouse strain in which all the three components of the Cd3 complex - Cd3ε, Cd3δ, and Cd3γ - are replaced by their human counterparts, CD3E, CD3D, and CD3G. Basic immunological assessments have confirmed that this strain of human CD3 EDG-replaced mice are entirely immune competent, and we have also demonstrated that a bispecific antibody that simultaneously binds to human CD3 and a tumor-associated antigen (e.g. ERBB2 or GPC3) can be evaluated in human CD3 EDG-replaced mice engrafted with tumors. Our mouse model provides a novel means to evaluate the in vivo efficacy of human CD3-mediated therapy.

Delayed formation of splenic pseudoaneurysm following nonoperative management in blunt splenic injury: multi-institutional study in Osaka, Japan.

BACKGROUND: Delayed rupture is well-known as a severe complication after splenic injury treated with nonoperative management (NOM). The incidence and timing of splenic pseudoaneurysm (SPA) formation, which is a cause of delayed rupture following splenic injury, have not been thoroughly investigated, and the timing of follow-up computed tomography (CT) is controversial. The objective of this study was to clarify the incidence and timing of both the delayed formation and spontaneous resolution of SPA following splenic injuries treated with NOM in several trauma centers in Japan. METHODS: This was a retrospective review of all patients with documented blunt splenic injury who were treated with NOM from 2003 through 2010 in five trauma and critical care centers. RESULTS: The present study consisted of 104 patients, including 16 patients (15.4%) with delayed formation of SPA (7 patients with Grade II and 9 with Grade III) during their clinical course. SPA was diagnosed with enhanced CT at a mean (SD) of 4.6 (2.1) hospital days (range, 1-8 days) after admission. Delayed formation of SPA was found in 30.4% of Grade II injuries and in 18.4% of Grade III injuries. Eight patients with delayed formation of SPA were observed without transcatheter arterial embolization during their entire stay. These SPAs were spontaneously occluded on follow-up enhanced CT or angiography. Spontaneous occlusion of SPA was confirmed at 5.2 (2.6) hospital days (range, 2-10 days) after diagnosis of delayed SPA. CONCLUSION: Delayed formation of SPAs was recognized with enhanced helical CT in 15% of all patients during hospital Days 1 to 8. About one half of the SPAs had occluded spontaneously without therapeutic intervention. Our results suggested that follow-up enhanced CT performed approximately 1 week after splenic injury may be useful to detect delayed SPA formation. LEVEL OF EVIDENCE: Epidemiologic study, level III.

Therapeutic potential of propagated hepatocyte transplantation in liver failure.

BACKGROUND: This study aimed to evaluate the therapeutic potential of intrasplenic transplantation of culture-propagated homologous hepatocytes in rats suffering from acute liver failure (ALF). METHODS: ALF was induced in dipeptidyl peptidase IV-negative (DPPIV(-)) Fischer 344 rats by totally removing the two anterior liver lobes (68% of the liver) and ligating the pedicle of the right lobe (24% of the liver). Hepatocytes isolated from DPPIV(+) Fischer 344 rats were cultured for 11 d to propagate 3-fold, and the resulting hepatocytes were dubbed "culture-propagated hepatocytes (CPHEPs)". A total of 1.5 × 10(7) cells of CPHEPs were transplanted intrasplenically before ALF induction (CPHEP group). Similarly, freshly isolated hepatocytes (FIHEPs) were transplanted as a positive control (FIHEP group), and culture medium (CM) was injected into rats as a negative control (CM group). RESULTS: The survival of the CPHEP group was comparable to that of the FIHEP group and longer than that of the CM group (P < 0.01). Both CPHEP and FIHEP transplantation improved blood parameters such as ammonia, total bilirubin, glutamic pyruvic transaminase, and glutamic oxaloacetic transaminase; transplantation also affected liver tissue parameters such as apoptosis rate and bromodeoxyuridine-labeling index. CONCLUSIONS: Transplantation of culture-propagated homologous hepatocytes has a remarkable therapeutic potential for ALF in rats.

A case of endometriosis of the appendix.

Endometriosis is prevalent among women of reproductive age, and is most commonly found in the gynecologic organs themselves and the surrounding pelvic peritoneum. Endometriosis of the appendix, however, is rare. Preoperative diagnosis is difficult and a definitive diagnosis is usually established following histopathological examination of the appendix. We report a case of endometriosis of the appendix in a 29-year-old woman who presented with right lower quadrant abdominal pain. Rebound tenderness was localized to McBurney's point. Her WBC count was 12,300/mm3 and her CRP was 6.497 mg/dl. Ultrasound and computed tomography detected a calcified region inside the cecum and slight thickening of the wall of the appendix. Based on these findings, the patient was diagnosed with acute appendicitis and underwent an appendectomy. The appendix appeared mildly congested, but the mucosa of the appendix was nearly normal and without macroscopic inflammation. Histopathological examination demonstrated ectopic endometrial glands and stroma in the muscularis. These stroma cells were positive for CD10 on immunohistochemical staining, establishing a diagnosis of endometriosis of the appendix. The patient had a good clinical course and no residual pain postoperatively.

Olfactory imprinting of amino acids in lacustrine sockeye salmon.

Juvenile salmon have an olfactory ability to imprint their natal stream odors, but neither the odor properties of natal stream water nor the imprinting timing and duration have been clarified as yet. Here we show, using electrophysiological and behavioral experiments, that one-year-old lacustrine sockeye salmon (Oncorhynchus nerka) can be imprinted around the stage of parr-smolt transformation (PST) by a single amino acid, 1 microM L-proline (Pro), or L-glutamic acid (Glu). We also show by real-time PCR that changes occur in mRNA levels of the salmon olfactory imprinting-related gene (SOIG) around PST. The electro-olfactogram (EOG) responses of test fish exposed to Pro in March (before PST) and April-June (during PST) for 2 weeks were significantly (1.7-fold) greater than those of non-exposed control fish, but not those of test fish exposed in July (after PST). When Pro and control water were added to the water inlets of a two-choice test tank during the spawning season 2 years after the test water exposure, 80% of maturing and matured test fish exposed before and during PST showed a preference for Pro, whereas those exposed after PST did not. The EOG response of test fish exposed to Pro or Glu for 1 hour, 6 hours, 1 day, 7 days, or 14 days in May revealed that only the response after 14 days of exposure was significantly (1.8-fold) greater than the control. The expression levels of SOIG mRNA increased before and during PST, and decreased after PST. We conclude that one-year-old lacustrine sockeye salmon can be imprinted by a single amino acid before and during PST, and that imprinting requires exposure for at least 14 days.

Growth and differentiation of colony-forming human hepatocytes in vitro.

BACKGROUND/AIMS: Parenchymal hepatocytes (PHs) of rat contain colony-forming parenchymal hepatocytes (CF-PHs) as a small fraction. We aimed to demonstrate the presence of CF-PHs in humans and characterize them with respect to growth and differentiation potential. METHODS: Human PHs were co-cultured with Swiss 3T3 cells in the medium containing human serum, EGF, nicontinamide, and ascorbic acid 2-phosphate. To examine differentiation potential hepatocytes were cultured on gels of Matrigel Matrix. RESULTS: Few PHs formed colonies, the colony-forming efficiency being as low as 0.01-0.09%. The CF-PHs could be subcultured up to 7 passages. They showed a liver epithelial cell-like morphology, and immunocytochemically positive for albumin (ALB), cytokeratin (CK) 7, 8, 18, and 19 in a pre- and early phase-confluence, whereas they showed a typical differentiated hepatocyte-like morphology, and positive for alpha(1)-antitrypsin, but negative for CK7 and 19 in condensed regions at confluence. The CF-PHs at late confluence expressed mRNAs of ALB, HNF4, and isoforms of cytochrome P450 at low levels. However, when cultured on Matrigel, these cells expressed them at high levels comparable to those of original PHs. CONCLUSIONS: We concluded that the human liver contains highly replicative hepatic progenitor-like cells as a minute population that retain a normal differentiation potential.

Efficient in vivo xenogeneic retroviral vector-mediated gene transduction into human hepatocytes.

We developed a method for efficient retroviral vector-mediated gene transfer into human hepatocytes, using a human hepatocyte-bearing mouse model. Normal human hepatocytes were transplanted into the livers of immunodeficient and liver-damaged mice. Donor hepatocytes multiplied and replaced the host hepatocytes, which yielded human hepatocyte-bearing mice (human hepatocyte-chimeric mice). As control cells, rat hepatocytes were similarly transplanted. The replacement level reached 86% at 8 weeks and 100% at 5 weeks posttransplantation of human and rat hepatocytes, respectively. Human and rat hepatocytes in the host liver showed a high bromodeoxyuridine-labeling index during the first 2 weeks posttransplantation. Human- and rat-chimeric mice were injected 7 and 10 days posttransplantation, respectively, with retroviral vectors carrying the beta-galactosidase gene and were thereafter injected daily for 20 and 10 days, respectively. The level of beta-galactosidase-positive hepatocytes in the human- and rat-chimeric mice reached 7.1 +/- 1.8% at 8 weeks and 5.3 +/- 0.9% at 5 weeks after transplantation, respectively. The human hepatocyte-chimeric mouse will be useful for testing the ability of vectors to transduce human cells.

Clinicopathologic features and prognostic factors of resected solitary small-sized hepatocellular carcinoma.

BACKGROUND/AIMS: Solitary small-sized HCCs tend to be considered as less aggressive cancer, and non-surgical treatments have recently been preferred. The aim of this study was to clarify the clinicopathological features and the prognostic factors of small-sized HCCs and to evaluate the significance of hepatic resection for them. METHODOLOGY: Eighty patients with HCC up to 2cm in diameter who had undergone hepatic resection were enrolled in this study. We investigated the clinicopathological features and evaluated the prognostic factors by univariate and multivariate analyses. RESULTS: The overall survival rates at 3, 5 and 10 years were 83%, 69% and 36%, respectively, and the corresponding disease-free survival rates were 63%, 41% and 10%. Well-differentiated, moderately differentiated and poorly differentiated HCC were detected in 29%, 65% and 6% of the patients, respectively. Furthermore, microscopic portal vein invasion (vp), hepatic vein invasion (vv) and intrahepatic metastasis (im) were positive in 15%, 4% and 10% of the patients, respectively. Multivariate analysis revealed that Child-Pugh classification (p=0.005) and vp (p=0.0008) were independent prognostic factors for survival rate and that platelet count (p=0.002) and tumor differentiation (p=0.0016) were independent prognostic factors for disease-free survival rate. CONCLUSIONS: Even solitary small-sized (up to 2cm in diameter) HCC already have the characteristics of advanced HCC. When hepatic function is well preserved, hepatic resection should be the first choice for local control, especially in cases of moderately to poorly differentiated HCC, because the frequency of microscopic vascular invasion is high.

Indication of hepatectomy for cirrhotic patients with hepatocellular carcinoma classified as Child-Pugh class B.

We clarified the indication of partial hepatectomy in hepatocellular carcinoma (HCC) patients with liver cirrhosis classified as Child-Pugh class B. Univariate analysis revealed that adverse prognostic factors were (1) the presence of ascites, (2) elevated total bilirubin (1.5 mg/dl or higher), (3) reduced choline esterase (160 IU/ or lower), (4) elevated alpha-fetoprotein (AFP) (400 ng/ml or higher), (5) microscopic vascular invasion, and (6) non-curative hepatectomy. Microvascular invasion was excluded in the multivariate analysis because this factor could not be predicted before hepatectomy. Multivariate analysis revealed that independent adverse prognostic factors were (1) elevated total bilirubin (1.5 mg/dl or higher), (2) presence of ascites, (3) elevated AFP (400 ng/ml or higher), and (4) non-curative hepatectomy. The overall 5-year survival rate of patients with none of or only one of the four adverse prognostic factors was 45.8%. The overall 5-year survival rate of patients with two or more adverse prognostic factors was only 7.0%. Partial hepatectomy is the first choice of treatment for patients with none of or only one of the four adverse prognostic factors, whereas orthotopic liver transplantation or other conservative treatment should be considered for patients with two or more adverse prognostic factors.

Indications of partial hepatectomy for transplantable hepatocellular carcinoma with compensated cirrhosis.

BACKGROUND: The appropriate treatment strategy for transplantable hepatocellular carcinoma (HCC) patients with compensated cirrhosis remains controversial. METHODS: Surgical outcomes were reviewed in 136 cirrhotic patients with transplantable HCC who had undergone partial hepatectomy. Transplantable HCC was defined as that corresponding to Milan's criteria. RESULTS: The adverse prognostic factors for both survival and disease-free survival were histologic surgical margin of 5 mm or less, Child-Pugh B, and the presence of hepatitis C virus infection. The overall 5-year survival and disease-free survival rates of patients with 1 or none of the adverse prognostic factors were 73% and 33%, respectively, whereas those of patients with 2 or 3 adverse prognostic factors were 36% and 17%, respectively. CONCLUSIONS: Transplantable HCC patients with 2 or 3 adverse prognostic factors should be considered candidates for liver transplantation, whereas patients with only 1 or none of the adverse prognostic factors are good candidates for partial hepatectomy.

Involvement of tumor necrosis factor-alpha receptor 1 and tumor necrosis factor-related apoptosis-inducing ligand-(TRAIL) receptor-2/DR-5, but not Fas, in graft injury in live-donor liver transplantation.

The pathway leading to cell death in clinical liver transplantation is not known. Eight liver transplant recipients and eight donors were enrolled in this study. Postoperative serum levels of alanine transferase had significantly increased in the recipients compared with those in the donors. Mild centri-lobular necrosis was observed in only liver tissues taken from the recipients. Tumor necrosis factor (TNF)-R1 and death receptor 5 expression levels had increased in liver tissues taken from the recipients. There were no changes in the levels of Fas/Fas ligand expression in liver tissues from either the donors or recipients. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression was down-regulated in donor liver after hepatectomy and liver allograft after implantation. The results suggest that, although ischemic liver injury was not serious, due to the short ischemia time, TNF and TRAIL signals are associated with liver ischemic injury in live-donor liver transplantation but Fas signal is not.

Near completely humanized liver in mice shows human-type metabolic responses to drugs.

Human hepatocytes were transplanted into urokinase-type plasminogen activator-transgenic SCID mice (uPA/SCID mice), which are immunodeficient and undergo liver failure. The transplanted cells were characterized in terms of their in vivo growth potential and functions. The human hepatocytes progressively repopulated the murine host liver. However, the recipients died when the replacement index (RI) of the human hepatocytes exceeded 50%. The hosts (chimeric mice) survived at RI >50% when treated with a drug that has anti-human complement factor activity, and these mice developed livers with RI values as high as 96%. In total, 36 chimeric mice were generated, and the rate of successful engraftment was as high as 92%. The yield of chimeric mice with RI >70% was 32%. The human hepatocytes in the murine host liver expressed mRNAs for a variety of human cytochrome P450 (hCYP) subtypes, in a manner that was similar to the donor liver. The mRNAs for hCYP3A4 and hCYP1A1/2 were induced in the liver in a CYP type-specific manner when the mice were treated with rifampicin and 3-methylcholanthrene, respectively. These results indicate that human hepatocytes that propagate in mice retain their normal pharmacological responses. We conclude that the chimeric mouse developed in the present study is a useful model for assessing the functions and pharmacological responses of human hepatocytes.

Gasless laparoscopic hepatic resection for cirrhotic patients with solid liver tumors.

Gasless laparoscopic hepatic resection with a 5-cm minilaparotomy was performed in 10 cirrhotic patients with small liver tumors. To maintain good visualization and working space during hepatic resections, we developed a simple retraction system. Mean operative time and blood loss were 291 minutes and 249 mL, respectively. No blood transfusion was required during the operations. No serious complications occurred such as gas embolism. Our laparoscopic procedures had various advantages. Blood, smoke, and water vapor could be aspirated by suction without disturbance of the visual field. There was no risk of gas embolism. It was possible to use conventional instruments through the ports or the wound made by a minilaparotomy. Hemostasis therefore could be performed easily. The procedure could be applicable to cirrhotic patients with some complications. This laparoscopic procedure is recommended for patients with small HCCs associated with liver cirrhosis who are not candidates for major hepatectomy.

Significance of the molar ratio of branched-chain amino acids to tyrosine before hepatectomy.

BACKGROUND/AIMS: To predict the degree of liver fibrosis and select a safe operative procedure, the correlation between liver fibrotic ratio and preoperative liver function variables were analyzed, and the significance of the molar ratio of branched-chain amino acids to tyrosine determined preoperatively was investigated. METHODOLOGY: Forty-four patients with hepatectomy were enrolled in this study. Liver tissue specimens excised from the patients were stained by Azan-Mallory's method. The liver fibrotic ratio was measured using an Image Cytometer. The correlation between liver fibrotic ratio and preoperative liver function values, including the serum level of branched-chain amino acids to tyrosine, were determined. RESULTS: The degrees of liver fibrosis (normal group, 1.4 +/- 0.7%; chronic hepatitis group, 4.5 +/- 2.8%; cirrhosis group, 8.4 +/- 2.4%) correlated significantly with preoperative liver function values such as branched-chain amino acids to tyrosine, hepaplastin test, type IV collagen 7s domain, total bilirubin, indocyanine green clearance retention rate at fifteen minutes, and platelet count. The serum level of branched-chain amino acids to tyrosine showed the most-significant correlation with the degree of liver fibrosis. CONCLUSIONS: The serum level of branched-chain amino acids to tyrosine correlates well with the degree of liver fibrosis, and this value is a useful preoperative parameter for predicting the degree of liver fibrosis and for selecting a safe operative procedure.