Takanori Sakaguchi

Claudins regulate the intestinal barrier in response to immune mediators.

BACKGROUND & AIMS To determine the functional role of immune mediators in the formation of the intestinal barrier, we have examined the regulation of claudin expression by interleukin (IL)-17 in human intestinal epithelial cells. METHODS Expression of claudins, extracellular signal-related (ERK) mitogen-activated protein kinases (MAPKs), and activated ERK MAPKs was determined by immunoblotting. Claudin membrane association was assessed by immunohistochemistry and claudin messenger RNA expression by Northern blot analysis. Intestinal epithelial barrier function was characterized through transepithelial electrical resistance and mannitol tracer flux. RESULTS IL-17 induced the development of a paracellular barrier of T84 cell monolayers. Inhibition of ERK activation with the MEK inhibitor PD98059 blocked IL-17 as well as basal development of tight junctions in T84 cells. IL-17 induced formation of tight junctions correlated with up-regulation of claudin-1 and claudin-2 gene transcription. Inhibition of MEK reduced the activated and basal expression of claudin-2 messenger RNA and protein expression. Functional MEK was required for the expression and membrane association of claudin-2 but not claudin-1 in T84 cells. CONCLUSIONS MEK activity is required for claudin-mediated formation of tight junctions. IL-17 is able to regulate the intestinal barrier through the ERK MAPK pathway.

Shigella flexneri regulates tight junction-associated proteins in human intestinal epithelial cells

Shigella spp. are a group of Gram‐negative enteric bacilli that cause acute dysentery in humans. We demonstrate that Shigella flexneri has evolved the ability to regulate functional components of tight junctions after interaction at the apical and basolateral pole of model intestinal epithelia. In the regulation of tight junctional protein assemblies, S. flexneri can engage serotype‐specific mechanisms, which targets not only expression, but also cellular distri‐bution and membrane association of components of tight junctions. Distinct mechanisms resulting in the regulation of tight junction‐associated proteins are initiated after either apical or basolateral interactions. S. flexneri serotype 2a has the ability to remove claudin‐1 from Triton X‐insoluble protein fractions upon apical exposure to T‐84 cell monolayers. S. flexneri serotype 2a and 5, but not the non‐invasive Escherichia coli strain F‐18, share the ability to regulate expression of ZO‐1, ZO‐2, E‐cadherin and to dephosphorylate occludin. The disruption of tight junctions is dependent on direct interaction of living Shigella with intestinal epithelial cells and is supported by heat‐stable secreted bacterial products. Intestinal epithelial cells have the ability to compensate in part for S. flexneri induced regulation of tight junction‐associated proteins.

Imaging mass spectrometry of gastric carcinoma in formalin‐fixed paraffin‐embedded tissue microarray

The popularity of imaging mass spectrometry (IMS) of tissue samples, which enables the direct scanning of tissue sections within a short time‐period, has been considerably increasing in cancer proteomics. Most pathological specimens stored in medical institutes are formalin‐fixed; thus, they had been regarded to be unsuitable for proteomic analyses, including IMS, until recently. Here, we report an easy‐to‐use screening method that enables the analysis of multiple samples in one experiment without extractions and purifications of proteins. We scanned, with an IMS technique, a tissue microarray (TMA) of formalin‐fixed paraffin‐embedded (FFPE) specimens. We detected a large amount of signals from trypsin‐treated FFPE‐TMA samples of gastric carcinoma tissues of different histological types. Of the signals detected, 54 were classified as signals specific to cancer with statistically significant differences between adenocarcinomas and normal tissues. We detected a total of 14 of the 54 signals as histological type‐specific with the support of statistical analyses. Tandem MS revealed that a signal specific to poorly differentiated cancer tissue corresponded to histone H4. Finally, we verified the IMS‐based finding by immunohistochemical analysis of more than 300 specimens spotted on TMAs; the immunoreactivity of histone H4 was remarkably strong in poorly differentiated cancer tissues. Thus, the application of IMS to FFPE‐TMA can enable high‐throughput analysis in cancer proteomics to aid in the understanding of molecular mechanisms underlying carcinogenesis, invasiveness, metastasis, and prognosis. Further, results obtained from the IMS of FFPE‐TMA can be readily confirmed by commonly used immunohistochemical analyses. (Cancer Sci 2009)

Clinicopathological prognostic factors and impact of surgical treatment of mass-forming intrahepatic cholangiocarcinoma.

The clinicopathological characteristics relevant to prognosis after surgical treatment of intrahepatic cholangiocarcinoma (ICC) remain unclear. In this study, the clinicopathological features of 19 patients with mass-forming ICC, the most common form of the disease, were reviewed to analyze prognostic determinants. Two or more segmentectomies of the liver with systematic lymphadenectomy were performed in 18 patients. Resection of the extrahepatic bile duct was performed in 14 patients, and reconstruction of the portal vein was accomplished in 5 patients. Stage IVA or IVB tumors were seen in 13 patients, and lymph node (LN) metastasis was present in 14 patients. The estimated 5-year survival rate after surgery for mass-forming ICC was 28%, with median survival time of 18 months. In univariate analysis, five variables were determined to be significantly correlated with poor survival of patients with mass-forming ICC after surgery. These variables include mass-forming ICC with periductal infiltration, perineural invasion, portal vein invasion, presence of intrahepatic metastasis, and two or more LN metastases. Survival rates of 5 patients without LN metastasis and 6 patients with a single LN metastasis were 80% and 33% at 5 years, respectively, while 8 patients with two or more LN metastasis failed to survive beyond 2 years. Multivariate analysis revealed the presence of intrahepatic metastasis to be an independent prognostic factor of poor survival. Hepatectomy with resection of the extrahepatic bile duct and systematic lymphadenectomy yields a good chance for prolonged survival for patients with mass-forming ICC when the lesion is singular and LN metastasis is limited to a regional LN. Because the presence of intrahepatic metastasis was closely related to a poor prognosis in patients with mass-forming ICC, efficacious chemotherapy would be needed to control development of the lesion.RésuméLes caractéristiques clinicopathologiques influençant le pronostic après traitement des cholangiocarcinomes intra-hépatiq.ues (CIH) ne sont pas claires. Dans cette étude, les caractéristiques clinicopathologiques chez 19 patients porteurs de CIH à forme tumorale, la forme la plus fréquente, ont été analysés à des fins pronostiques. On a réalisé une segmentectomie de deux segments ou plus avec lymphadénectomie systématique chez 18 patients, une résection des voies biliaires extra-hépatiques chez 14 et une reconstruction de la veine porte chez 5 patients. Treize patients avaient une tumeur stade IVA ou IVB; 14 avaient des métastases ganglionnaires. La survie à 5 ans après chirurgie pour CIH à forme tumorale a été de 28%; la médiane de survie a été de 18 mois. En analyse univariée, on a trouvé cinq variables significativement associées à une survie médiocre chez les patients opérés de CIH à forme tumorale. Ces variables sont un CIH avec infiltration péricanulaire, un envahissement perineural, un envahissement portai, la présence de métastases hépatiques, et des métastases de deux ganglions ou plus. La survie de cinq patients sans métastase ganglionnaire et de six patients avec une seule métastase ganglionnaire ont été, respectivement, de 80% et de 33% à 5 ans, alors qu’aucun des huit patients avec deux métastases ganglionnaires ou plus n’a survécu au-delà de deux ans. En analyse multivariée, la présence de métastases intrahépatiques était un facteur indépendant de mauvais pronostic. Une hépatectomie avec résection des voies biliaires extra-hépatiques associée à un curage lymphatique systématique améliore les chances de survie prolongée en cas de CIH à forme tumorale lorsque la lésion est unique et les métastases ganglionnaires sont limitées à un seul ganglion lymphatique régional. Puisque la présence de métastases intrahépatiques est étroitement en rapport avec un mauvais pronostic chez les patients porteurs de CIH à forme tumorale, une chimiothérapie efficace est nécessaire pour contrôler l’évolution.ResumenTras el tratamiento quirúrgico, las características clínicopatológicas pronósticas más importantes para los pacientes con colangiocarcinomas intrahepáticos (ICC) son poco conocidas. En este estudio se revisan las características clínicopatológicas más frecuentes en 19 pacientes con grandes tumores ICC, con objeto de determinar los factores pronósticos más importantes. 18 casos fueron tratados mediante dos o más segmentectomías hepáticas y linfadenectomía sistemática. En 14 pacientes se procedió a la resección y subsiguiente reconstrucción de la vía biliar extrahepática y en 5 de la vena porta. 13 pacientes pertenecían al estadio IV A o IV B y adenopatias metastásicas (LN) se registraron en 14 enfermos. Tras el acto quirúrgico el porcentaje medio estimado de supervivencia a los 5 años fue del 28%, con un tiempo de supervivencia de 18 meses. En pacientes con ICC que cursan con una tumoración macroscópicamente visible y palpable, el análisis univariante detectó 5 variables significativas por lo que a la escasa supervivencia se refiere: tumoración ICC con infiltración periductal, invasión perineural o de la vena porta, existencia de metástasis intrahepáticas y 2 o más adenopatías (LN) metastásicas. La supervivencia a los 5 años de 5 pacientes sin metástasis ganglionares (LN) y con una sola adenopatía metastásica fue del 80% y 33%, mientras que 8 pacientes con dos o más adenopatias metastásicas (LN) no sobrevivieron más de 2 años. El análisis multivariante demostró que las metástasis intrahepáticas constituyen un factor pronóstico independiente, de escasa supervivencia. La hepatectomía con resección de la vía biliar extrahepática asociada a una sistemática linfadenectomia puede, con suerte, prolongar la supervivencia de pacientes con tumores ICC palpables, cuando la tumoración es única y las adenopatias metastásicas son exclusivamente regionales. Dado que la presencia de metástasis intrahepáticas es signo de mal pronóstico, se precisa una eficaz quimioterapia para controlar el desarrollo de este tumor.

Interleukin-2 Receptor β Subunit-dependent and -independent Regulation of Intestinal Epithelial Tight Junctions

Interleukin (IL)-15 is able to regulate tight junction formation in intestinal epithelial cells. However, the mechanisms that regulate the intestinal barrier function in response to IL-15 and the involved subunits of the IL-15 ligand-receptor system are unknown. We determined the IL-2Rβ subunit and IL-15-dependent regulation of tight junction-associated proteins in the human intestinal epithelial cell line T-84. The IL-2Rβ subunit was expressed and induced signal transduction in caveolin enriched rafts in intestinal epithelial cells. IL-15-mediated tightening of intestinal epithelial monolayers correlated with the enhanced recruitment of tight junction proteins into Triton X-100-insoluble protein fractions. IL-15-mediated up-regulation of ZO-1 and ZO-2 expression was independent of the IL-2Rβ subunit, whereas the phosphorylation of occludin and enhanced membrane association of claudin-1 and claudin-2 by IL-15 required the presence of the IL-2Rβ subunit. Recruitment of claudins and hyperphosphorylated occludin into tight junctions resulted in a more marked induction of tight junction formation in intestinal epithelial cells than the up-regulation of ZO-1 and ZO-2 by itself. The regulation of the intestinal epithelial barrier function by IL-15 involves IL-2Rβ-dependent and -independent signaling pathways leading to the recruitment of claudins, hyperphosphorylated occludin, ZO-1, and ZO-2 into the tight junctional protein complex.

Flux of the l-Serine Metabolism in Rabbit, Human, and Dog Livers SUBSTANTIAL CONTRIBUTIONS OF BOTH MITOCHONDRIAL AND PEROXISOMAL SERINE:PYRUVATE/ALANINE:GLYOXYLATE AMINOTRANSFERASE

l-Serine metabolism in rabbit, dog, and human livers was investigated, focusing on the relative contributions of the three pathways, one initiated by serine dehydratase, another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Under quasi-physiological in vitro conditions (1 mm l-serine and 0.25 mmpyruvate), flux through serine dehydratase accounted for only traces, and that through SPT/AGT substantially contributed no matter whether the enzyme was located in peroxisomes (rabbit and human) or largely in mitochondria (dog). As for flux through serine hydroxymethyltransferase and GCS, the conversion of serine to glycine occurred fairly rapidly, followed by GCS-mediated slow decarboxylation of the accumulated glycine. The flux through GCS was relatively high in the dog and low in the rabbit, and only in the dog was it comparable with that through SPT/AGT. An in vivo experiment withl-[3-3H,14C]serine as the substrate indicated that in rabbit liver, gluconeogenesis froml-serine proceeds mainly via hydroxypyruvate. Because an important role in the conversion of glyoxylate to glycine has been assigned to peroxisomal SPT/AGT from the studies on primary hyperoxaluria type 1, these results suggest that SPT/AGT in this organelle plays dual roles in the metabolism of glyoxylate and serine.

Analysis of anatomic variants of mesenteric veins by 3-dimensional portography using multidetector-row computed tomography

BACKGROUND It is important to be aware of mesenteric venous variants to perform peripancreatic surgery. We investigated the usefulness of 3-dimensional (3-D) portography. METHODS Vessels were reconstructed using computer software in 102 patients undergoing multidetector-row computed tomography (MDCT) scheduled for gastrointestinal or hepatobiliary-pancreatic surgery. RESULTS The superior mesenteric vein (SMV) was composed of single and double trunks around the splenoportal confluence in 78 and 24 patients, respectively. The inferior mesenteric vein joined the splenic vein (68.5%), SMV (18.5%), and splenoportal confluence (7.6%). The left gastric vein joined the splenic vein (46.3%), portal vein (39.0%), and splenoportal confluence (14.7%). Seventy-nine patients showed a gastrocolic trunk, mostly composed of the right gastroepiploic vein and veins from the colonic hepatic flexure. Intraoperative findings were identical to 3-D diagnosis in 68 gastrectomized and 9 pancreatectomized patients. CONCLUSION Although mesenteric venous tributaries are complex, 3-D portography is helpful for surgeons to safely perform peripancreatic surgery.

Lysophosphatidylcholine acyltransferase 1 altered phospholipid composition and regulated hepatoma progression.

BACKGROUND & AIMS Several lipid synthesis pathways play important roles in the development and progression of hepatocellular carcinoma (HCC), although the precise molecular mechanisms remain to be elucidated. Here, we show the relationship between HCC progression and alteration of phospholipid composition regulated by lysophosphatidylcholine acyltransferase (LPCAT). METHODS Molecular lipidomic screening was performed by imaging mass spectrometry (IMS) in 37 resected HCC specimens. RT-PCR and Western blotting were carried out to examine the mRNA and protein levels of LPCATs, which catalyze the conversion of lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) and have substrate specificity for some kinds of fatty acids. We examined the effect of LPCAT1 overexpression or knockdown on cell proliferation, migration, and invasion in HCC cell lines. RESULTS IMS revealed the increase of PC species with palmitoleic acid or oleic acid at the sn-2-position and the reduction of LPC with palmitic acid at the sn-1-position in HCC tissues. mRNA and protein of LPCAT1, responsible for LPC to PC conversion, were more abundant in HCCs than in the surrounding parenchyma. In cell line experiments, LPCAT1 overexpression enriched PCs observed in IMS and promoted cell proliferation, migration, and invasion. LPCAT1 knockdown did viceversa. CONCLUSIONS Enrichment or depletion of some specific PCs, was found in HCC by IMS. Alteration of phospholipid composition in HCC would affect tumor character. LPCAT1 modulates phospholipid composition to create favorable conditions to HCC cells. LPCAT1 is a potent target molecule to inhibit HCC progression.

Flux of the l-Serine Metabolism in Rat Liver THE PREDOMINANT CONTRIBUTION OF SERINE DEHYDRATASE

l-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by l-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mm l-serine and 0.25 mm pyruvate) was about 1 10 of that through SDH. Flux through GCS accounted for only several percent of the amount ofl-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from l-serine were evaluatedin vivo based on the principle that 3H at the 3 position of l-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10–20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of l-serine in rat liver.

Alteration of reticuloendothelial phagocytic function and tumor necrosis factor-α production after total hepatic ischemia

BACKGROUND This study was conducted to determine whether the duration of total hepatic ischemia influences reticuloendothelial phagocytic activity and tumor necrosis factor (TNF)-alpha production after reperfusion. METHODS Male rats pretreated with either normal saline (NS group) or gadolinium chloride (7 mg/kg) for 2 days to inhibit Kupffer cell function (GC group) were subjected to 30, 60, or 90 min of total hepatic ischemia. RESULTS The animals tolerated hepatic ischemia well for 30 and 60 min. Although the 7-day survival rate of the NS group decreased to 28% after 90 min of hepatic ischemia, that of the GC group improved significantly to 68% (P<0.01). In the NS group, plasma alanine transaminase and TNF-alpha levels after reperfusion increased with the length of hepatic ischemia. The phagocytic index (PI) after 60 min of reperfusion following 90 min of hepatic ischemia showed significant depression compared with the preischemic level and the value after 30 or 60 min of ischemia. The GC group had significantly lower plasma alanine transaminase and TNF-alpha levels as well as significantly less polymorphonuclear leukocyte infiltration in the liver compared with the NS group. The preischemic PI was significantly inhibited in the GC group when compared with that in the NS group, but PI in the GC group did not change significantly after reperfusion, irrespective of the ischemic time. CONCLUSIONS This study demonstrated that warm ischemia of up to 60 min is tolerable for normal rat liver without a detrimental effect on phagocytic activity. Modulation of Kupffer cell function may have the potential to prevent reperfusion injury after hepatic ischemia, which may allow safe prolongation of the ischemic time.