Hirokazu Tsuji

Attenuated liver fibrosis and depressed serum albumin levels in carbon tetrachloride-treated IL-6-deficient mice.

Chronic intermittent injection of carbon tetrachloride (CCI4) for more than 10 weeks induced liver fibrosis in mice, as evidenced by positive Azan staining and increased intrahepatic collagen content. Preceding the onset of liver fibrosis, interleukin‐6 (IL‐6) gene expression was enhanced in liver and immunoreactive IL‐6 was detected in infiltrating inflammatory cells. To delineate the role of IL‐6 in this process, we treated IL‐6‐deficient mice with CCl4 in a similar manner for 12 weeks, after which fibrotic changes were less evident and serum albumin levels were lower in IL‐6‐deficient than wild‐type mice. Moreover, CCl4‐induced expression of transforming growth factor β1 and hepatocyte growth factor genes in liver was significantly reduced in IL‐6‐deficient mice. Thus, IL‐6 may be vitally involved in fibrotic changes and maintenance of serum albumin levels, partly by modulating intrahepatic expression of these cytokines. J. Leukoc. Biol. 66: 601–608; 1999.

Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma

Human telomerase reverse transcriptase, hTERT, has been identified as the catalytic enzyme required for telomere elongation. hTERT is expressed in most tumor cells but seldom expressed in most human adult cells. It has been reported that 80% to 90% of hepatocellular carcinomas (HCCs) express hTERT, making the enzyme a potential target in immunotherapy for HCC. In the current study, we identified hTERT‐derived, HLA‐A*2402–restricted cytotoxic T cell (CTL) epitopes and analyzed hTERT‐specific CTL responses in patients with HCC. Peptides containing the epitopes showed high affinity to bind HLA‐A*2402 in a major histocompatibility complex binding assay and were able to induce hTERT‐specific CTLs in both hTERT cDNA‐immunized HLA‐A*2402/Kb transgenic mice and patients with HCC. The CTLs were able to kill hepatoma cell lines depending on hTERT expression levels in an HLA‐A*2402–restricted manner and induced irrespective of hepatitis viral infection. The number of single hTERT epitope‐specific T cells detected by ELISPOT assay was 10 to 100 specific cells per 3 × 105 PBMCs, and positive T cell responses were observed in 6.9% to 12.5% of HCC patients. hTERT‐specific T cell responses were observed even in the patients with early stages of HCC. The frequency of hTERT/tetramer+CD8+ T cells in the tumor tissue of patients with HCC was quite high, and they were functional. In conclusion, these results suggest that hTERT is an attractive target for T‐cell–based immunotherapy for HCC, and the identified hTERT epitopes may be valuable both for immunotherapy and for analyzing host immune responses to HCC. (HEPATOLOGY 2006;43: 1284–1294.)

Identification of α‐fetoprotein‐derived peptides recognized by cytotoxic T lymphocytes in HLA‐A24+ patients with hepatocellular carcinoma

α‐Fetoprotein (AFP) has been proposed as a potential target forT‐cell‐based immunotherapy for hepatocellular carcinoma (HCC), but the number of its epitopes that have been identified is limited and the status of AFP‐specific immunological responses in HCC patients has not been well‐characterized. To address the issue, we examined the possibility of inducing AFP‐specific cytotoxic T cells (CTLs) using novel HLA‐A*2402‐restricted T‐cell epitopes (HLA, human leukocyte antigen) derived from AFP and then analyzed the relationship between its frequency of occurrence and clinical features associated with patients having HCC. Five AFP‐derived peptides containing HLA‐A*2402 binding motifs and showing high binding affinity to HLA‐A*2402 induced CTLs to produce IFN‐γ and kill an AFP‐producing hepatoma cell line. The frequency of AFP‐specific CTLs was 30–190 per 1 × 106 peripheral blood mononuclear cells, which was the same as that of other immunogenic cancer associated antigen‐derived epitopes. Analyses of the relationships between AFP‐specific CTL responses and clinical features of patients with HCC revealed that AFP epitopes were more frequently recognized by CTLs in patients with advanced HCC correlating to tumor factors or the stage of TNM classification. The analyses of CTL responses before and after HCC treatments showed that the treatments changed the frequency of AFP‐specific CTLs. In conclusion, we identified five HLA‐A*2402‐restrictedT‐cell epitopes derived from AFP. The newly identified AFP epitopes could be a valuable component of HCC immunotherapy and for analyzing host immune responses to HCC.

Possible association of Bifidobacterium and Lactobacillus in the gut microbiota of patients with major depressive disorder

BACKGROUNDnBifidobacterium and Lactobacillus in the gut have been suggested to have a beneficial effect on stress response and depressive disorder. We examined whether these bacterial counts are reduced in patients with major depressive disorder (MDD) than in healthy controls.nnnMETHODnBifidobacterium and Lactobacillus counts in fecal samples were estimated in 43 patients and 57 controls using bacterial rRNA-targeted reverse transcription-quantitative polymerase chain reactionnnnRESULTSnThe patients had significantly lower Bifidobacterium counts (P=0.012) and tended to have lower Lactobacillus counts (P=0.067) than the controls. Individuals whose bacterial counts below the optimal cut-off point (9.53 and 6.49log10 cells/g for Bifidobacterium and Lactobacillus, respectively) were significantly more common in the patients than in the controls for both bacteria (Bifidobacterium: odds ratio 3.23, 95% confidence interval [CI] 1.38-7.54, P=0.010; Lactobacillus: 2.57, 95% CI 1.14-5.78, P=0.027). Using the same cut-off points, we observed an association between the bacterial counts and Irritable bowel syndrome. Frequency of fermented milk consumption was associated with higher Bifidobacterium counts in the patients.nnnLIMITATIONSnThe findings should be interpreted with caution since effects of gender and diet were not fully taken into account in the analysis.nnnCONCLUSIONnOur results provide direct evidence, for the first time, that individuals with lower Bifidobacterium and/or Lactobacillus counts are more common in patients with MDD compared to controls. Our findings provide new insight into the pathophysiology of MDD and will enhance future research on the use of pro- and prebiotics in the treatment of MDD.

Combined therapy of transcatheter hepatic arterial embolization with intratumoral dendritic cell infusion for hepatocellular carcinoma: clinical safety

The curative treatments for hepatocellular carcinoma (HCC), including surgical resection and radiofrequency ablation (RFA), do not prevent tumour recurrence effectively. Dendritic cell (DC)‐based immunotherapies are believed to contribute to the eradication of the residual and recurrent tumour cells. The current study was designed to assess the safety and bioactivity of DC infusion into tumour tissues following transcatheter hepatic arterial embolization (TAE) for patients with cirrhosis and HCC. Peripheral blood mononuclear cells (PBMCs) were differentiated into phenotypically confirmed DCs. Ten patients were administered autologous DCs through an arterial catheter during TAE treatment. Shortly thereafter, some HCC nodules were treated additionally to achieve the curative local therapeutic effects. There was no clinical or serological evidence of adverse events, including hepatic failure or autoimmune responses in any patients, in addition to those due to TAE. Following the infusion of 111Indium‐labelled DCs, DCs were detectable inside and around the HCC nodules for up to 17u2003days, and were associated with lymphocyte and monocyte infiltration. Interestingly, T lymphocyte responses were induced against peptides derived from the tumour antigens, Her‐2/neu, MRP3, hTERT and AFP, 4 weeks after the infusion in some patients. The cumulative survival rates were not significantly changed by this strategy. These results demonstrate that transcatheter arterial DC infusion into tumour tissues following TAE treatment is feasible and safe for patients with cirrhosis and HCC. Furthermore, the antigen‐non‐specific, immature DC infusion may induce immune responses to unprimed tumour antigens, providing a plausible strategy to enhance tumour immunity.

Sensitive Quantitative Analysis of the Meconium Bacterial Microbiota in Healthy Term Infants Born Vaginally or by Cesarean Section.

For decades, babies were thought to be born germ-free, but recent evidences suggest that they are already exposed to various bacteria in utero. However, the data on population levels of such pioneer gut bacteria, particularly in context to birth mode, is sparse. We herein aimed to quantify such bacteria from the meconium of 151 healthy term Japanese infants born vaginally or by C-section. Neonatal first meconium was obtained within 24–48 h of delivery; RNA was extracted and subjected to reverse-transcription-quantitative PCR using specific primers for Clostridium coccoides group, C. leptum subgroup, Bacteroides fragilis group, Atopobium cluster, Prevotella, Bifidobacterium, Lactobacillus, Enterococcus, Enterobacteriaceae, Staphylococcus, Enterococcus, Streptococcus, C. perfringens, and C. difficile. We detected several bacterial groups in both vaginally- and cesarean-born infants. B. fragilis group, Enterobacteriaceae, Enterococcus, Streptococcus, and Staphylococcus were detected in more than 50% of infants, with counts ranging from 105 to 108 cells/g sample. About 30–35% samples harbored Bifidobacterium and Lactobacillus (104–105 cells/g); whereas C. coccoides group, C. leptum subgroup and C. perfringens were detected in 10–20% infants (103–105 cells/g). Compared to vaginally-born babies, cesarean-born babies were significantly less often colonized with Lactobacillus genus (6% vs. 37%; P = 0.01) and Lactobacillus gasseri subgroup (6% vs. 31%; P = 0.04). Overall, seven Lactobacillus subgroups/species, i.e., L. gasseri subgroup, L. ruminis subgroup, L. casei subgroup, L. reuteri subgroup, L. sakei subgroup, L. plantarum subgroup, and L. brevis were detected in the samples from vaginally-born group, whereas only two members, i.e., L. gasseri subgroup and L. brevis were detected in the cesarean group. These data corroborate that several bacterial clades may already be present before birth in term infants’ gut. Further, lower detection rate of lactobacilli in cesarean-born babies suggests that the primary source of lactobacilli in infant gut is mainly from maternal vaginal and–to a lesser extent–anal microbiota during vaginal delivery, and that the colonization by some important Lactobacillus species is delayed in babies delivered via cesarean-section.

Diversity of Intestinal Clostridium coccoides Group in the Japanese Population, as Demonstrated by Reverse Transcription-Quantitative PCR

We used sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) to quantify the Clostridium coccoides group, which is a major anaerobic population in the human intestine. For this purpose, the C. coccoides group was classified into 3 subgroups and 19 species for expediency in accordance with the existing database, and specific primers were newly developed to evaluate them. Population levels of the C. coccoides group in human feces determined by RT-qPCR were equivalent to those determined by fluorescence in situ hybridization. RT-qPCR analysis of fecal samples from 96 volunteers (32 young children, 32 adults and 32 elderly) by using the 22 new primer sets together with the C. coccoides group-specific primer setm revealed that (i) total counts obtained as the sum of the 3 subgroups and 19 species were equivalent to the results obtained by using the C. coccoides group-specific primer set; (ii) total C. coccoides-group counts in the elderly were significantly lower than those in young children and adults; (iii) genus Blautia was the most common subgroup in the human intestinal C. coccoides-group populations at all age populations tested; (iv) the prevalences of Fusicatenibacter saccharivorans and genus Dorea were significantly higher in adults than in young children and the elderly; and (v) the prevalences of C. scindens and C. hylemonae, both of which produce secondary bile acid in the human intestine, were significantly higher in the elderly than in young children and adults. Hierarchical clustering and principal component analysis showed clear separation of the bacterial components between adult and elderly populations. Taken together, these data suggest that aging plays an important role in the diversity of C. coccoides-group populations in human intestinal microbiota; changes in this diversity likely influence the health of the host.

The development and clinical features of splenic aneurysm associated with liver cirrhosis.

Abstract: Objectives: Splenic artery aneurysm (SAA) is usually asymptomatic, but can be fatal if it ruptures. Portal hypertensive patients with varix or splenomegaly are sometimes complicated by SAA. However, there have been no large‐scale clinical studies regarding whether liver cirrhosis itself is associated with splenic aneurysm regardless of varix or splenomegaly.

Transgenic mice aberrantly expressing pyruvate dehydrogenase complex E2 component on biliary epithelial cells do not show primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disorder that specifically destroys biliary epithelial cells (BECs). In patients with PBC, the immunodominant pyruvate dehydrogenase complex E2 component (PDC‐E2), identified as an antigen for disease‐specific anti‐mitochondrial antibody, is expressed aberrantly in the BEC cytoplasm. The present study focused on the pathophysiological role of aberrant PDC‐E2 in the development of PBC. The BEC‐specific cytokeratin‐19 promoter and PDC‐E2 gene were cloned from a mouse cDNA library. The constructed transgene was microinjected into fertilized eggs of mice, and the offspring were identified by Southern blotting and reverse transcriptase–polymerase chain reaction. The protein expression was confirmed by immunoprecipitation, immunoblotting and immunohistochemical staining. Five founder lines were identified as carrying the PDC‐E2 gene, and one of these lines expressed PDC‐E2 mRNA. The protein expression of exogenous PDC‐E2 was detected in the liver. The transgenic mouse line showed diffuse expression of PDC‐E2 in the BEC cytoplasm. Biochemical, serological and histological features of PBC were not detected. We established transgenic mice that constitutively express PDC‐E2. The results indicated that aberrant PDC‐E2 expression in the cytoplasm of BECs is not sufficient for the initiation of autoimmunity. Additional factors may be required to establish a model of PBC.

Participation of Endogenously Produced Interferon gamma in Interleukin 4-Mediated Tumor Rejection

To elucidate the molecular mechanism underlying IL-4-induced tumor rejection, we challenged mice with a mouse adenocarcinoma cell line, colon 26, genetically engineered to express constitutively IL-4 gene (colon 26/IL-4). Immunocompetent BALB/c mice rejected colon 26/IL-4 cells but not parental cells or cells transduced with a control gene (colon 26/control). Moreover, on rechallenge, parental cells and colon 26/control cells were rejected by normal BALB/c mice that had previously rejected colon 26/IL-4. However, both nude and severe combined immunodeficiency (SCID) mice failed to reject colon 26/IL-4 as well as parental or colon 26/control cells. In contrast, nude mice did reject colon 26/IL-4 after transfer of lymphocytes obtained from the draining lymph nodes of BALB/c mice injected with colon 26/IL-4. These results indicate that challenging mice with colon 26/IL-4 tumor cells resulted in the generation of memory cytotoxic T lymphocytes in the draining lymph nodes. At 3 days after the challenge, IFN-gamma, IL-12 p35, and p40 mRNA expression was selectively enhanced in the draining lymph nodes of mice bearing colon 26/IL-4 cells. Finally, mice deficient in the IFN-gamma gene did not reject colon 26/IL-4 cells. These results suggest that IL-4-induced memory cytotoxic T lymphocyte generation requires IFN-gamma production in the draining lymph nodes, in order to generate a protective immune response.