Masahiro Kido

Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells.

Carbohydrate chains on a glycoprotein are important not only for protein conformation, transport and stability, but also for cell–cell and cell–matrix interactions. UDP‐Gal:N‐acetylglucosamine β‐1,4‐galactosyltransferase (GalT) (EC 2.4.1.38) is the enzyme which transfers galactose (Gal) to the terminal N‐acetylglucosamine (GlcNAc) of complex‐type N‐glycans in the Golgi apparatus. In addition, it has also been suggested that this enzyme is involved directly in cell–cell interactions during fertilization and early embryogenesis through a subpopulation of this enzyme distributed on the cell surface. In this study, GalT‐deficient mice were produced by gene targeting in order to examine the pathological effects of the deficiency. GalT‐deficient mice were born normally and were fertile, but they exhibited growth retardation and semi‐lethality. Epithelial cell proliferation of the skin and small intestine was enhanced, and cell differentiation in intestinal villi was abnormal. These observations suggest that GalT plays critical roles in the regulation of proliferation and differentiation of epithelial cells after birth, although this enzyme is dispensable during embryonic development.

Clinical Application of Serum Pepsinogen I and II Levels for Mass Screening to Detect Gastric Cancer

A considerable number of gastric cancers derive from stomach mucosa where chronic atrophic gastritis is severe and extensive. Based on the fact that the serum pepsinogen levels provide a precise measure of the extent of chronic atrophic gastritis, we have devised a mass screening method involving serum pepsinogen measurement to identify subjects at high risk of gastric cancer. In 1991, we screened 4,647 workers (male: 4,113, female: 534, mean age: 49.0 years) at a Japanese company using this method. Out of 875 subjects (18.8%) with a serum pepsinogen I level of less than 50 μg/liter and a pepsinogen I/II ratio of less than 3.0, 676 subjects (14.5%) were selected for further investigation by endoscopy. This led to the detection of four subjects (0.086%) with gastric cancer (three in an early stage) and four subjects with adenoma. The cancer detection rate of this new screening method was comparable, and in some respects superior, to that of the traditional barium X‐ray screening. Since the incidence of test‐positive subjects was as low as 10% amongst subjects aged less than 40, this screening method appears to be especially useful for screening of younger generations. The new method is less expensive than the traditional barium X‐ray and subjects experience little discomfort. Further, many serum samples can be quickly measured simultaneously. The results of this study have indicated that serum pepsinogen screening provides a valuable method for detecting gastric cancers.

Fatal Autoimmune Hepatitis Induced by Concurrent Loss of Naturally Arising Regulatory T Cells and PD-1-Mediated Signaling

BACKGROUND & AIMS Because of the lack of animal models developing spontaneous autoimmune hepatitis (AIH), the molecular mechanisms involved in the development of AIH are still unclear. This study aims to examine the regulatory roles of naturally arising CD4(+)CD25(+) regulatory T (Treg) cells and programmed cell death 1 (PD-1)-mediated signaling in the development of AIH. METHODS To induce a concurrent loss of Treg cells and PD-1-mediated signaling, neonatal thymectomy (NTx), which severely reduces the number of Treg cells, was performed on PD-1(-/-) mice. After the NTx, we performed histologic examination, assessed autoantibody production and infiltrating cells in the liver, and conducted adoptive transfer experiments. RESULTS In contrast to NTx mice and PD-1(-/-) mice, NTx-PD-1(-/-) mice produced antinuclear antibodies and developed fatal hepatitis characterized by a CD4(+) and CD8(+) T-cell infiltration invading the parenchyma with massive lobular necrosis. Induction of AIH in NTx-PD-1(-/-) mice was suppressed by transfer of Treg cells, even derived from PD-1(-/-) mice. Transfer of total but not CD4(+) T-cell-depleted splenocytes from NTx-PD-1(-/-) mice into RAG2(-/-) mice induced the development of severe hepatitis. In contrast, the transfer of CD8(+) T-cell-depleted splenocytes triggered only mononuclear infiltrates without massive necrosis of the parenchyma. CONCLUSIONS NTx-PD-1(-/-) mice are the first mouse model of spontaneous fatal AIH. The concurrent loss of Treg cells and PD-1-mediated signaling can induce the development of fatal AIH. Autoreactive CD4(+) T cells are essential for induction of AIH, whereas CD8(+) T cells play an important role in progression to fatal hepatic damage.

Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2‐polarizing conditions

Background Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP.

Reductive Surgery Plus Percutaneous Isolated Hepatic Perfusion for Multiple Advanced Hepatocellular Carcinoma

Objective:To evaluate the efficacy of a novel 2-stage treatment with reductive surgery plus percutaneous isolated hepatic perfusion (PIHP) for multiple hepatocellular carcinoma (HCC), which was previously unresectable. Summary Background Data:Surgical resection is the treatment of choice for HCC, but the majority of patients with advanced HCC are not suitable candidates. PIHP is a minimally invasive surgery that allows high-dose regional chemotherapy of the liver, and our phase II studies have shown its profound efficacy for the local control of advanced HCC. Methods:Twenty-five patients with multiple advanced HCC were enrolled in this prospective study. In the first stage, all patients underwent reductive hepatectomy: major hepatectomy in 13 patients and segmentectomy or less in 12. In 2 patients with subsegmentectomy, the retropancreatic and periportal metastatic lymph nodes were synchronously resected. Regardless of the type of hepatectomy, all patients routinely underwent cholecystectomy, and ligations of the right gastric artery and arterial collaterals of the remnant liver to increase the safety and efficacy of PIHP. In the second stage, PIHP with doxorubicin 60–120 mg/m2/treatment was planned for a period of 1 to 3 months after surgery. Results:Of 25 enrolled patients, 22 successfully underwent PIHP an average of 1.8 times for the local control of residual liver tumors. In the remaining 3 patients, PIHP was abandoned because 2 had rapid disease progression and 1 had liver failure after surgery. In 22 patients with the 2-stage treatment, 19 (86%) had objective local tumor control (10 complete remissions and 9 partial responses with a median response duration of 16 months). The actuarial survival rate of all 25 patients was 42% at 5 years. Conclusions:Reductive surgery plus PIHP produced a strong antitumoral effect on multiple advanced HCC, when liver function allows this concentrated treatment approach, and offers long-term survival in a subset of patients who were previously deemed to have unresectable disease.

Dysregulated Generation of Follicular Helper T Cells in the Spleen Triggers Fatal Autoimmune Hepatitis in Mice

BACKGROUND & AIMS To clarify mechanisms involved in the development of autoimmune hepatitis (AIH), we recently developed a mouse model of spontaneous AIH by inducing a concurrent loss of Foxp3(+) regulatory T cells and programmed cell death 1 (PD-1)-mediated signaling. Fatal AIH in these mice was characterized by severe T-cell infiltration and huge production of antinuclear antibodies (Abs). This study aims to identify induction sites, responsible T-cell subsets, and key molecules for induction of AIH. METHODS To develop the mouse model of AIH, neonatal thymectomy (NTx) was performed on PD-1-deficient (PD-1(-/-)) mice. We then conducted neonatal splenectomy or in vivo administration of Abs to cytokines, chemokines, or cell-surface molecules. RESULTS In NTx-PD-1(-/-) mice, either neonatal splenectomy or in vivo CD4(+) T-cell depletion suppressed CD4(+) and CD8(+) T-cell infiltration in the liver. In the induction phase of AIH, splenic CD4(+) T cells were localized in B-cell follicles with huge germinal centers and showed the Bcl6(+) inducible costimulator (ICOS)(+) interleukin (IL)-21(+) IL-21 receptor (IL-21R)(+) follicular helper T (T(FH)) cell phenotype. Blocking Abs to ICOS or IL-21 suppressed T(FH)-cell generation and induction of AIH. In addition, IL-21 produced by T(FH) cells drove CD8(+) T-cell activation. Splenic T(FH) cells and CD8(+) T cells expressed CCR6, and CCL20 expression was elevated in the liver. Administration of anti-CCL20 suppressed migration of these T cells to the liver and induction of AIH. CONCLUSIONS Dysregulated T(FH) cells in the spleen are responsible for the induction of fatal AIH, and CCR6-CCL20 axis-dependent migration of splenic T cells is crucial to induce AIH in NTx-PD-1(-/-) mice.

Significant role of middle hepatic vein in remnant liver regeneration of right-lobe living donors

For adult patients with end-stage liver disease, living-donor liver transplantation (LDLT) of right-lobe grafts with or without the middle hepatic vein (MHV) has been increasingly used in recent years. We investigated the role of the MHV in donor remnant liver regeneration after right-lobe LDLT, which has not been described in previous studies. A total of eight living donors were included in this study of right-lobe LDLT. Four donors underwent right lobectomy (without MHV), and the remaining four underwent extended right lobectomy (with MHV). Regeneration of the donor remnant liver was assessed by volumetric computed tomography studies before and 90 days after LDLT. Comparison between the right-lobe and extended right-lobe donors did not show a clear-cut difference in the net increase of remnant liver volume at 3 months. However, the mean volume increase of the medial segment at the 90th postoperative day was 7% in the extended right-lobe donors and 61% in the right-lobe donors, showing a lower value in the remnant livers without MHV. The MHV plays a specific role in remnant liver regeneration of right-lobe living donors. We expect that this knowledge will contribute to securing a margin of safety in right-lobe LDLT.

Helicobacter pylori Promotes the Production of Thymic Stromal Lymphopoietin by Gastric Epithelial Cells and Induces Dendritic Cell-Mediated Inflammatory Th2 Responses

ABSTRACT Helicobacter pylori colonizes the stomach and induces strong, specific local and systemic humoral and cell-mediated immunity, resulting in the development of chronic gastritis in humans. Although H. pylori-induced chronic atrophic gastritis is characterized by marked infiltration of T helper type 1 (Th1) cytokine-producing CD4+ T cells, almost all of the inflamed gastric mucosae also contain focal lymphoid aggregates with germinal centers. In addition, typical H. pylori-induced chronic gastritis in children, called follicular gastritis, is characterized by B-cell follicle formation in the gastric mucosa. The aim of this study was to examine whether thymic stromal lymphopoietin (TSLP), an epithelial-cell-derived cytokine inducing a dendritic cell (DC)-mediated inflammatory Th2 response, is involved in Th2 responses triggering B-cell activation in H. pylori-induced gastritis. Here, we show that H. pylori triggered human gastric epithelial cells to produce TSLP, together with the DC-attracting chemokine MIP-3α and the B-cell-activating factor BAFF. After DCs were incubated with supernatants from H. pylori-infected epithelial cells, the conditioned cells expressed high levels of costimulatory molecules, such as CD80, and triggered naïve CD4+ T cells to produce high levels of the Th2 cytokines interleukin-4 and interleukin-13 and of the inflammatory cytokines tumor necrosis factor alpha and gamma interferon. In contrast, after incubation of the supernatants with the neutralizing antibodies to TSLP, the conditioned DCs did not prime T cells to produce high levels of Th2 cytokines. These results, together with the finding that TSLP was expressed by the epithelial cells of human follicular gastritis, suggest that H. pylori can directly trigger epithelial cells to produce TSLP. It also suggests that TSLP-mediated DC activation may be involved in Th2 responses triggering B-cell activation in H. pylori-induced gastritis.

Human TSLP directly enhances expansion of CD8+ T cells

Human thymic stromal lymphopoietin (TSLP) promotes CD4+ T‐cell proliferation both directly and indirectly through dendritic cell (DC) activation. Although human TSLP‐activated DCs induce CD8+ T‐cell proliferation, it is not clear whether TSLP acts directly on CD8+ T cells. In this study, we show that human CD8+ T cells activated by T‐cell receptor stimulation expressed TSLP receptor (TSLPR), and that TSLP directly enhanced proliferation of activated CD8+ T cells. Although non‐stimulated human CD8+ T cells from peripheral blood did not express TSLPR, CD8+ T cells activated by anti‐CD3 plus anti‐CD28 did express TSLPR. After T‐cell receptor stimulation, TSLP directly enhanced the expansion of activated CD8+ T cells. Interestingly, using monocyte‐derived DCs pulsed with a cytomegalovirus (CMV)‐specific pp65 peptide, we found that although interleukin‐2 allowed expansion of both CMV‐specific and non‐specific CD8+ T cells, TSLP induced expansion of only CMV‐specific CD8+ T cells. These results suggest that human TSLP directly enhances expansion of CD8+ T cells and that the direct and indirect action of TSLP on expansion of target antigen‐specific CD8+ T cells may be beneficial to adoptive cell transfer immunotherapy.

Progression of autoimmune hepatitis is mediated by IL-18-producing dendritic cells and hepatic CXCL9 expression in mice

Clinical manifestations of autoimmune hepatitis (AIH) range from mild chronic to acute, sometimes fulminant hepatitis. However, it is unknown how the progression to fatal hepatitis occurs. We developed a mouse model of fatal AIH by inducing a concurrent loss of forkhead box P3+ regulatory T cells and programmed cell death‐1 (PD‐1)‐mediated signaling. In this model, dysregulated follicular helper T cells in the spleen are responsible for the induction, and the C‐C chemokine receptor 6/C‐C chemokine ligand 20 axis is crucial for the migration of these T cells into the liver. Using this fatal AIH model, we aimed to clarify key molecules triggering fatal AIH progression. During progression, T‐bet together with interferon (IFN)‐γ and C‐X‐C chemokine receptor (CXCR)3 were highly expressed in the inflamed liver, suggesting helper T (Th)1‐type inflammation. T cells that dominantly expanded in the spleen and the inflamed liver were CXCR3‐expressing CD8+ T cells; depletion of these CD8+ T cells suppressed AIH progression. Expression of one CXCR3 ligand, chemokine (C‐X‐C motif) ligand (CXCL)9, was elevated in the liver. CXCL9‐expressing macrophages/Kupffer cells were colocalized with infiltrating T cells, and in vivo administration of anti‐CXCL9 suppressed AIH progression. In addition, serum levels of interleukin (IL)‐18, but not IL‐1β, were elevated during progression, and dendritic cells in the spleen and liver highly produced IL‐18. In vivo administration of anti‐IL‐18R suppressed the increase of splenic CXCR3+ T cells and the progression to fatal AIH. Moreover, tumor necrosis factor alpha, but not IFN‐γ, was involved in up‐regulating CXCL9 in the liver and for increased serum levels of IL‐18. Conclusion: These data suggest that, in our mouse model, fatal progression of AIH is mediated by IL‐18‐dependent differentiation of T cells into Th1 cells and effector T cells, respectively, and that CXCR3‐CXCL9 axis‐dependent migration of those T cells is crucial for fatal progression. (Hepatology 2014;60:224–236)