Hepatocytes and Endothelial Networks in a Fluid-Based In Vitro Model of Liver Drug Metabolism.

Drug-induced liver toxicity remains a major cause of drug withdrawal from animal testing and human clinical trials. A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. Previously reported fluid-based systems have some advantages for hepatocyte culture, but have insufficient liver-specific functions because they simply involve moving conventional hepatocyte cultures from a dish into a fluid-based system. Importantly, these cultures have no liver tissue-specific structures that construct liver-specific cellular polarities, such as apical, basolateral, and basal faces. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions. Impact statement A functional liver culture model corresponding to the liver is urgently required; however, in previous liver models, it has proven difficult to stably maintain multiple liver functions. In this study, we developed a fluid-based system for our liver tissue culture models. The liver tissues that were constructed in our originally designed fluid-based systems represent a tissue culture model for studying hepatic functions. Together, our findings show that by mimicking the structure of the liver in the body, our system effectively maintains multiple liver-specific functions.

Microliter-ordered automatic blood sampling system for fully quantitative analysis of small-animal PET.

OBJECTIVE: The objective of the present study was to develop a fully automated blood sampling system for kinetic analysis in mice positron emission tomography (PET) studies. Quantitative PET imaging requires radioactivity concentrations in arterial plasma to estimate the behavior of an administered radiopharmaceutical in target organs. Conventional manual blood sampling has several drawbacks, such as the need for troubleshooting in regard to blood collection, necessary personnel, and the radiation exposure dose. We recently developed and verified the operability of a fully automated blood sampling system (automatic blood dispensing system-ABDS). Here, we report the results of fully quantitative measurements of the cerebral metabolic rate of glucose (CMRglc) in mice using the ABDS. METHODS: Under 1% isoflurane anesthesia, a catheter was inserted into the femoral artery of nine wild-type male mice. Immediately after injection of 18F-fluorodeoxyglucose (FDG) (13.2 ± 3.93 MBq in 0.1 mL saline), arterial blood samples were drawn using the ABDS and then analyzed using CD-Well, a system we previously developed that can measure radioactivity concentration (Bq/µL) using a few microliters of blood in the plasma and whole blood separately. In total, 16 blood samplings were conducted in 60 min as follows: 10 s × 9; 70 s × 2; 120 s × 1; 250 s × 1; 10 min × 2; and 30 min × 1. Dynamic PET scans were conducted concurrently using a small-animal PET/computed tomography (CT) (PET/CT) scanner. Full kinetics modeling using a two-tissue-three-compartment model was applied to calculate CMRglc. Blood volume was also estimated. RESULTS: No significant differences were observed between the manual and ABDS measurements. A proportional error was detected only for plasma. The mean ± standard deviation CMRglc value in the mice was 5.43 ± 1.98 mg/100 g/min (30.2 ± 11 µmol/min/100 g), consistent with a previous report. CONCLUSIONS: The automated microliter-ordered blood sampling system developed in the present study appears to be useful for absolute quantification of CMRglc in mice PET studies.

A case of gastric adenocarcinoma of fundic gland type resected by combination of laparoscopic and endoscopic approaches to neoplasia with non-exposure technique (CLEAN-NET).

A male in his eighties attended our hospital for further evaluation of gastric cancer. A gastroscopy revealed a whitish flat elevated lesion (Paris, 0-IIa) of 15 mm in diameter on the greater curvature of the proximal fornix. The preoperative diagnosis was intra-mucosal differentiated gastric cancer, and a novel therapeutic approach, combination of laparoscopic and endoscopic approaches to neoplasia with non-exposure technique (CLEAN-NET) was applied and the lesion was resected in a single piece without any complications. Histopathological findings revealed atypical glandular epithelium proliferated in the mucosa and shallow layer (300 µm) of submucosa. These cells stained positive for pepsinogen-I and the final diagnosis was gastric cancer of fundic gland type (GAFT). There was no lymph-vascular involvement and free horizontal and vertical margins were confirmed. CLEAN-NET could be a therapeutic option for GAFT at low risk of lymph node metastasis because it prevents excess wall defect and exposure of cancer cells into the peritoneal cavity.

[A case of metastatic colon cancer effectively treated by XELOX and IRIS].

The patient was a 78-year-old woman who suffered from right upper quadrant pain. She was diagnosed as colon cancer with hepatic metastasis. Initial chemotherapy using capecitabine plus oxaliplatin(XELOX)+bevacizumab achieved partial response. After 12 months, XELOX was discontinued due to Grade 3 hand-foot syndrome and peripheral neuropathy. Irinotecan plus oral S-1(a combination of tegafur, 5-chloro-2, 4-dihydroxypyridine, and potassium oxonate)and(IRIS)+bevacizumab were continued as second-line therapy, and her status was maintained as stable disease. XELOX and IRIS regimens do not require catheter placement and long infusion process, providing a great advantage to patients.

Acetaminophen-induced hepatotoxicity in a liver tissue model consisting of primary hepatocytes assembling around an endothelial cell network.

Primary hepatocytes have been used in drug development for the evaluation of hepatotoxicity of candidate compounds. However, the rapid depression of their hepatic characters in vitro must be improved to predict toxicity with higher accuracy. We have hypothesized that a well organized tissue construct that includes nonparenchymal cells and appropriate scaffold material(s) could overcome this difficulty by remediating the viability and physiological function of primary hepatocytes. In this study, we constructed an in vitro liver tissue model, consisting of mouse primary hepatocytes assembling around an endothelial cell network on Engelbreth-Holm-Swarm gel, and examined its response to acetaminophen treatment. The increase in lactate dehydrogenase release after the exposure to acetaminophen was induced earlier in the liver tissue model than in monolayer hepatocytes alone, suggesting that the tissue model was more sensitive to an acetaminophen-induced toxicity. On the basis of our results, we conclude that liver tissue models of this kind may enhance the responses of hepatocytes against xenobiotics via the maintenance of hepatic genes and functions such as cytochrome P450s. These findings will contribute to the development of more accurate systems for evaluating hepatotoxicity.

Butter feeding enhances TNF-alpha production from macrophages and lymphocyte adherence in murine small intestinal microvessels.

BACKGROUND AND AIM: Dietary fat is known to modulate immune functions. Intake of an animal fat-rich diet has been linked to increased risk of inflammation; however, little is known about how animal fat ingestion directly affects intestinal immune function. The objective of this study was to assess the effect of butter feeding on lymphocyte migration in intestinal mucosa and the changes in adhesion molecules and cytokines involved in this effect. METHODS: T-lymphocytes isolated from the spleen were fluorescence-labeled and injected into recipient mice. Butter was administered into the duodenum, and villus microvessels of the small intestinal mucosa were observed under an intravital microscope. mRNA expression of adhesion molecules and cytokines in the intestinal mucosa were determined by quantitative PCR. The effect of butter feeding on tumor necrosis factor (TNF)-alpha mRNA expression of intestinal macrophages was also determined. RESULTS: Intraluminal butter administration significantly increased lymphocyte adherence to intestinal microvessels accompanied by increases in expression levels of adhesion molecules ICAM-1, MAdCAM-1 and VCAM-1. This accumulation was significantly attenuated by anti-MAdCAM-1 and anti-ICAM-1 antibodies. Butter administration significantly increased TNF-alpha in the lamina proprial macrophages but not interleukin-6. Anti-TNF-alpha treatment attenuated the enhanced expression of adhesion molecules induced by butter administration. CONCLUSION: T-lymphocyte adherence to microvessels of the small intestinal mucosa was significantly enhanced after butter ingestion. This enhancement is due to increase in expression levels of adhesion molecules of the intestinal mucosa, which is mediated by TNF-alpha from macrophages in the intestinal lamina propria.

CXCL12 and CCL20 play a significant role in mucosal T-lymphocyte adherence to intestinal microvessels in mice.

OBJECTIVE: Although it is known that the chemokines CXCL12 and CCL20 are expressed in the intestine, their contribution to lymphocyte homing has not been investigated in detail. The authors investigated whether the CXCL12-CXCR4 and CCL20-CCR6 systems are involved in T lymphocyte-endothelial interaction in microvessels of the small and large intestines. METHODS: Labeled lamina proprial lymphocytes (LPLs) were administered to mice, and their adhesion to microvessels of normal and TNF-alpha -induced inflamed intestinal mucosa was observed under an intravital microscope. Antibodies against CXCL12, CCL-20, or CCL-25 were administered prior to lymphocyte administration, and in some experiments CXCR4 or CCR6 on LPLs was desensitized with an excess amount of chemokine. RESULTS: LPLs adhered to microvessels of the ileum and colon, and TNF-alpha induced a significant accumulation at both sites. Blocking of the CXCL12-CXCR4 system significantly inhibited the LPL adhesion in the ileum and colon under both normal and TNF-alpha -treated conditions. However, blocking of the CCL20-CCR6 system significantly attenuated LPL adhesion only under a TNF-alpha -treated condition. There was an additive inhibitory effect on LPL adherence by CXCL12 and CCL20 blocking in TNF-alpha -induced inflamed intestines. There was also an additive function of the CCL25-CCR9 system in LPL accumulation in the small intestine. CONCLUSION: Several chemokine systems may play significant roles cooperatively in vivo in LPL adherence to microvessels of intestinal mucosa.

[A case of fulminant hepatitis E treated with artificial liver support].

A 40-year-old man, who had suffered from general malaise and brown urine during his stay in China, was admitted with remarkable jaundice and hepatocellular disorders soon after he returned to Japan. Because his coagulation test results worsened, he was transferred to our hospital. No evidence of hepatitis A-D virus infection, autoimmune hepatitis, or metabolic disorders was noticed. His prothrombin time was extended (18%), grade II encephalopathy appeared on the second hospital day, and fulminant hepatitis was diagnosed. Artificial liver support was introduced, and his hepatic coma and coagulation parameters gradually recovered. Genotype IV hepatitis E virus RNA was detected in his early phase sera and also both IgG and IgM type anti-hepatitis E virus antibodies were detected. Fulminant hepatitis E resulting from infection in China was diagnosed.

Demonstration of functional role of TECK/CCL25 in T lymphocyte-endothelium interaction in inflamed and uninflamed intestinal mucosa.

It has recently been suggested that C-C chemokines may play a role in the organ-specific homing of lymphocytes, but there is not enough in vivo evidence in intestinal mucosa. The aim of this study was to examine whether thymus-expressed chemokine (TECK)/CCL25 and its ligand CCR9 are involved in T-lymphocyte interaction with microvessels of murine intestinal mucosa. T lymphocytes from the small intestine were fluorescence labeled, and their adhesion to mucosal microvessels was observed by intravital microscopy. Lamina proprial lymphocytes (LPL) and intraepithelial lymphocytes (IEL) adhered to both the small intestine and colon, and desensitization of CCR9 with TECK/CCL25 or anti-TECK/CCL25 antibody significantly inhibited these adhesions only in small intestine. At both sites, TNF-alpha significantly increased LPL adhesion but not IEL adhesion. Desensitization of CCR9 or anti-TECK/CCL25 antibody also attenuated the TNF-alpha-induced LPL adhesion in the small intestine. Increased expression of TECK/CCL25 by TNF-alpha was observed in the lamina propria of small intestine. TECK/CCL25 may thus play an important role in the adherence of mucosal lymphocytes to the microvessels of the small intestine but not the colon under uninflamed as well as inflamed conditions.