Assessment of colonic contents in patients with chronic constipation using MRI.

Although chronic constipation is common, colonic functional evaluating tests are uncommon. This study examines whether chronic constipation and gastrointestinal symptoms are correlated with the lateral diameter of the colon measured from MRI images. We included chronic constipation patients in a prospective, cross-sectional study using MRI at three centers. We divided 3D MRI colorectal images into 6 segments using with specified sequences and selected the maximum luminal diameter from each segment. We used the GSRS questionnaire to evaluate gastrointestinal symptoms. We evaluated the correlation between luminal diameters and GSRS scores. We found the following positive correlations: descending colon and unsatisfactory defecation symptoms; sigmoid colon and diarrhea; and rectum and constipation. The sum and ratio of the ascending and sigmoid colon diameters correlated with nausea and diarrhea. The sum of the transvers to the sigmoid colon diameter also correlated with nausea and diarrhea. The sum of all segment diameters correlated with nausea and constipation. In conclusion, we showed cross-sectional study of colonic MRI correlate with gastrointestinal symptoms. MRI might be useful for colonic motility evaluations to determine appropriate constipation treatments (Clinical trial registry number UMIN 000021274).

In vivo immunological antitumor effect of OK-432-stimulated dendritic cell transfer after radiofrequency ablation.

Radiofrequency ablation therapy (RFA) is a radical treatment for liver cancers and induces tumor antigen-specific immune responses. In the present study, we examined the antitumor effects of focal OK-432-stimulated dendritic cell (DC) transfer combined with RFA and analyzed the functional mechanisms involved using a murine model. C57BL/6 mice were injected subcutaneously with colon cancer cells (MC38) in their bilateral flanks. After the establishment of tumors, the subcutaneous tumor on one flank was treated using RFA, and then OK-432-stimulated DCs were injected locally. The antitumor effect of the treatment was evaluated by measuring the size of the tumor on the opposite flank, and the immunological responses were assessed using tumor-infiltrating lymphocytes, splenocytes and draining lymph nodes. Tumor growth was strongly inhibited in mice that exhibited efficient DC migration after RFA and OK-432-stimulated DC transfer, as compared to mice treated with RFA alone or treatment involving immature DC transfer. We also demonstrated that the antitumor effect of this treatment depended on both CD8-positive and CD4-positive cells. On the basis of our findings, we believe that combination therapy for metastatic liver cancer consisting of OK-432-stimulated DCs in combination with RFA can proceed to clinical trials, and it is anticipated to be markedly superior to RFA single therapy.

Genetic polymorphism in cyclooxygenase-2 promoter affects hepatic inflammation and fibrosis in patients with chronic hepatitis C.

Cyclooxygenase (COX)-2 is involved in inflammation, anti-apoptosis and carcinogenesis. The -1195GG genotype of single nucleotide polymorphism (SNP) in COX-2 promoter was associated with low platelet counts in patients with chronic hepatitis C. Polymorphism of patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene (rs738409 C>G) have been reported to be associated with cirrhosis, and the major genotype of SNPs near interleukin (IL)28B are related to viral clearance. The present study was designed to assess the contribution of these SNPs to disease progression in patients with chronic hepatitis C. The study enrolled 220 Japanese patients with chronic hepatitis C. Three SNPs, -1195 COX-2, PNPLA3 and IL28B (rs8099917), were genotyped in order to analyze their association with hepatic fibrosis and inflammation. The -1195GG genotype in COX-2 was associated with advanced fibrosis and higher levels of inflammation in the liver tissues. The major genotype of IL28B was also associated with advanced fibrosis, but the polymorphism of PNPLA3 was neither associated with fibrosis nor inflammation. Multivariate analysis showed that -1195GG in COX-2 is an independent factor associated with advanced fibrosis, while the major genotype of IL28B and HCV genotype 2 were other independent factors. In conclusion, the -1195GG genotype in COX-2 is a genetic marker for liver disease progression, while the PNPLA3 genotypes are not associated with disease progression in Japanese patients with chronic hepatitis C.

Reactivation of Epstein-Barr virus in B cells of patients with chronic hepatitis C.

Hepatitis C virus (HCV) infection is associated with lymphoproliferative disorders. HCV infection of B cells is a predictive factor for lymphoproliferative disorders in patients with chronic hepatitis C, although its molecular mechanisms remain unknown. Epstein-Barr virus (EBV) is a B cell-tropic virus with the potential to cause lymphoproliferative disorders, and its reactivation is induced by several viruses and cytokines. The possibility that HCV infection triggers reactivation of EBV and induces lymphoproliferative disorders were investigated. Expression of EBV mRNAs was analyzed by RT-PCR in patients infected with HCV and control subjects, and correlations between reactivation of EBV and markers for lymphoproliferative disorders were investigated. BZLF1 mRNA, a starter molecule of reactivation, was detected in peripheral blood mononuclear cells from 12 of 52 (23%), patients infected with HCV and the frequency was higher than in healthy subjects [3 of 43 (9%), P = 0.032]. But the presence of the BZLF1 mRNA was not associated with an abnormality of markers for lymphoproliferative disorders. This study on BZLF1 mRNA expression among lymphoid cell subsets showed that reactivation of EBV was observed specifically in B cells. The BZLF1 mRNA disappeared following anti-viral therapy and remained negative after eradication of HCV in patients with a sustained viral response, while the EBER1 RNA, a marker for persistence of EBV, was detected throughout the therapy. Infection with HCV induces reactivation of EBV in B cells, but this reactivation was not associated directly with lymphoproliferative disorders triggered by HCV.

Immune response of cytotoxic T lymphocytes and possibility of vaccine development for hepatitis C virus infection.

Immune responses of cytotoxic T lymphocytes (CTLs) are implicated in viral eradication and the pathogenesis of hepatitis C. Weak CTL response against hepatitis C virus (HCV) may lead to a persistent infection. HCV infection impairs the function of HCV-specific CTLs; HCV proteins are thought to actively suppress host immune responses, including CTLs. Induction of a strong HCV-specific CTL response in HCV-infected patients can facilitate complete HCV clearance. Thus, the development of a vaccine that can induce potent CTL response against HCV is strongly expected. We investigated HCV-specific CTL responses by enzyme-linked immuno-spot assay and/or synthetic peptides and identified over 40 novel CTL epitopes in the HCV protein. Our findings may contribute to the development of the HCV vaccine. In this paper, we describe the CTL responses in HCV infection and the attempts at vaccine development based on recent scientific articles.

Infection of B cells with hepatitis C virus for the development of lymphoproliferative disorders in patients with chronic hepatitis C.

Infection with hepatitis C virus (HCV) is associated with lymphoproliferative disorders, represented by essential mixed cryoglobulinemia and B-cell non-Hodgkin's lymphoma, but the pathogenic mechanism remains obscure. HCV may infect B cells or interact with their cell surface receptors, and induce lymphoproliferation. The influence of HCV infection of B cells on the development of lymphoproliferative disorders was evaluated in 75 patients with persistent HCV infection. HCV infection was more prevalent (63% vs. 16%, 14%, or 17% P < 0.05 for each), and HCV RNA levels were higher (3.35 +/- 3.85 vs. 1.75 +/- 2.52, 2.15 +/- 2.94 or 2.10 +/- 2.90 log copies/100 ng, P < 0.01 for each) in B cells than CD4(+), CD8(+) T cells or other cells. Negative-strand HCV RNA, as a marker of viral replication, was detected in B cells from four of the 75 (5%) patients. Markers for lymphoproliferative disorders were more frequent in the 50 patients with chronic hepatitis C than the 32 with chronic hepatitis B, including cryoglobulinemia (26% vs. 0%, P < 0.001), low CH(50) levels (48% vs. 3%, P = 0.012), and the clonality of B cells (12% vs. 0%, P < 0.01). By multivariate analysis, HCV RNA in B cells was an independent factor associated with the presence of at least one marker for lymphoproliferation (odds ratio: 1.98 [95% confidence interval: 1.36-7.24], P = 0.027). Based on the results obtained, the infection of B cells with HCV would play an important role in the development of lymphoproliferative disorders.

Magnitude of CD8 T-cell responses against hepatitis C virus and severity of hepatitis do not necessarily determine outcomes in acute hepatitis C virus infection.

AIM: We investigated the relationship between the magnitude of comprehensive hepatitis C virus (HCV)-specific CD8(+) T-cell responses and the clinical course of acute HCV infection. METHODS: Six consecutive patients with acute HCV infection were studied. Analysis of HCV-specific CD8(+) T-cell responses was performed using an interferon-gamma-based enzyme-linked immunospot assay using peripheral CD8(+) T-cells, monocytes and 297 20-mer synthetic peptides overlapping by 10 residues and spanning the entire HCV sequence of genotype 1b. RESULTS: Five patients presented detectable HCV-specific CD8(+) T-cell responses against a single and different peptide, whereas 1 patient showed responses against three different peptides. Neither the magnitude of HCV-specific CD8(+) T-cell responses nor the severity of hepatitis predicts the outcome of acute hepatitis. The maximum number of HCV-specific CD8(+) T-cells correlated with maximum serum alanine aminotransferase level during the course (r = 0.841, P = 0.036). CONCLUSIONS: HCV-specific CD8(+) T-cell responses were detectable in all 6 patients with acute HCV infection, and 6 novel HCV-specific CTL epitopes were identified. Acute HCV infection can resolve with detectable HCV-specific CD8(+) T-cell responses, but without development of antibody against HCV.

Dendritic cells stimulated with cytidine-phosphate-guanosine oligodeoxynucleotides and interferon-alpha-expressing tumor cells effectively reduce outgrowth of established tumors in vivo.

Dendritic cells (DC) are potent antigen-presenting cells that elicit immune responses to foreign antigens. We have previously demonstrated the synergistic effects of cytidine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODN) and interferon (IFN)-alpha on DC maturation in vitro. In the present study, the antitumor effects of DC preincubated with IFN-alpha gene-overexpressing murine colorectal cancer MC38 cells (MC38-IFN-alpha) and CpG ODN were evaluated in a poorly immunogenic murine cancer system. When we injected DC preincubated with MC38-IFNalpha and CpG ODN subcutaneously to mice bearing MC38 wild-type tumors, the outgrowth of the established parental tumors was suppressed significantly compared with that following administration of DC with MC38-IFN-alpha (P = 0.008). All mice injected with DC preincubated with MC38-IFN-alpha and CpG ODN rejected a subsequent parental tumor challenge. Immunohistochemical and flow cytometric analyses showed that CD4(+), CD8(+), and NK1.1(+) cells markedly infiltrated the established tumors of mice treated with DC preincubated with MC38-IFN-alpha and CpG ODN. From the results in immune cell-depleted mice, CD4(+) and asialo-GM-1(+) cells seemed to contribute to the antitumor effects induced by the combination DC therapy. Furthermore, non-specific cytolysis was detected when splenocytes of mice inoculated with DC preincubated with MC38-IFNalpha and CpG ODN were used as effector cells. Using an interleukin (IL)-12-neutralizing antibody it was suggested that IL-12 stimulates natural killer cells and contributes in part to the antitumor effects induced by DC incubated with CpG ODN and IFN-alpha. As DC-based immunotherapy with CpG ODN and IFN-alpha-expressing tumor cells induces a potent antitumor immune response, it should be considered for clinical application.

Intrahepatic status of regulatory T cells in autoimmune liver diseases and chronic viral hepatitis.

AIM: Regulatory T cells (Tregs) maintain immunological tolerance and suppress autoreactive immune responses. We evaluated the intrahepatic status of Tregs in patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), chronic hepatitis C (CH-C), or chronic hepatitis B (CH-B). METHODS: We analyzed 85 patients (20 AIH, 22 PBC, 27 CH-C, and 16 CH-B) and 14 controls. Using liver tissue samples obtained by needle biopsy or from marginal parts of resected metastatic liver tumors in the controls, immunohistochemical analyses of forkhead box P3(+), which is a specific marker for Tregs, CD4(+), and CD8(+) cells were performed. RESULTS: Intrahepatic Tregs were significantly more infiltrated in patients with liver diseases than in the controls. There were significantly fewer intrahepatic Tregs in the AIH patients than in the PBC patients (P = 0.037). Patients with alow frequency of intrahepatic Tregs were detected significantly more in the AIH and CH-B groups than in the PBC and CH-C groups (P < 0.05). In addition, the frequency of Tregs decreased in the liver of PBC patients as the pathological stage of the disease advanced. We found significantly less infiltration of CD4(+) T cells in AIH than in other diseases (P < 0.05). Liver-infiltrating CD8(+) T cells were detected more frequently in the CH-B group than in other groups (P < 0.003). CONCLUSION: Intrahepatic Tregs were increased in both patients with autoimmune liver diseases and those with viral hepatitis. In autoimmune liver diseases, intrahepatic Tregs were fewer in the AIH patients than in the PBC patients.

Immune responses in hepatitis C virus infection and mechanisms of hepatitis C virus persistence.

Immune responses against hepatitis C virus (HCV) play a crucial role in the pathogenesis of chronic hepatitis C. HCV infection often persists and leads to chronic hepatitis and eventually cirrhosis. Accumulated data suggest that HCV proteins suppress host immune responses through the suppression of functions of immune cells, such as cytotoxic T lymphocytes, natural killer cells, and dendritic cells. They also suppress the type 1 interferon signaling system. The resulting insufficient immune responses against HCV lead to the sustained infection. The appropriate control of immune responses would contribute to the eradication of HCV and the improvement of hepatitis, but there are still many issues to be clarified. This review describes the scientific evidence to support these emerging concepts, and will touch on the implications for improving antiviral therapy.

Cytidine-phosphate-guanosine oligodeoxynucleotides and interferon-alpha-expressing tumor cells effectively induce dendritic cell maturation in vitro.

BACKGROUND: Dendritic cells (DCs) play an important role in immune response and cytidine-phosphate-guanosine (CpG) oligodeoxynucleotides (ODN) as well as interferon (IFN)-alpha have been proven to induce DC maturation. In this study, the synergistic effects of CpG-ODN and IFN-alpha on DC maturation were evaluated. MATERIALS AND METHODS: Surface molecules on DCs and the stimulatory responses of DCs to allogeneic splenocytes were analyzed after cultivation with CpG-ODN and IFN-alpha-overexpressing murine colorectal cancer MC38 cells (MC38-IFNalpha). RESULTS: Co-incubation with CpG-ODN and MC38-IFNalpha, but not wild-type MC38 cells (MC38-WT), effectively up-regulated co-stimulatory molecules on the DCs. CpG, in combination with IFN-alpha, stimulated IL-1beta and TNF-alpha production by DCs effectively. When DCs preincubated with CpG-ODN and MC38-IFNalpha were co-incubated with allogeneic splenocytes in vitro, the proliferation of these splenocytes was significantly enhanced compared with that of splenocytes incubated with CpG-ODN and MC38-WT cells (p = 0.041). CONCLUSION: Since CpG-ODN and IFN-alpha have synergistic effects on DC maturation, they may induce potent antitumor immune responses and combination therapy should be considered for clinical application.

Immune responses of liver-infiltrating lymphocytes and peripheral blood mononuclear cells to hepatitis C virus core and NS3 antigens.

BACKGROUND AND AIM: Th1/Th2 cytokine balance is thought to play an important role in antiviral immunity and pathogenesis in viral infection. Ex vivo hepatitis C virus (HCV) antigen-specific T-cell responses were investigated. METHODS: Using enzyme-linked immunospot assay, HCV core and NS3 antigen-specific interferon-gamma-, interleukin-4- and interleukin-10-secreting cells were enumerated in peripheral blood mononuclear cells (PBMCs) from 30 chronic hepatitis C patients and 16 healthy controls, and in liver-infiltrating lymphocytes (LILs) from 17 of the 30 patients. RESULTS: IFN-gamma- and IL-10-secreting cells in response to stimulation with HCV core and NS3 antigen were detectable in both PBMCs and LILs from patients with chronic hepatitis C, although frequencies of the cytokine-secreting cells were much higher in LILs than PBMCs. They were not detectable in PBMCs of healthy controls except for IL-10-secreting cells in response to HCV NS3 antigen stimulation. IL-4-secreting cells were hardly detectable in both PBMC and LIL in both the patients and the healthy controls. Frequencies of HCV NS3 antigen-specific IFN-gamma- and IL-10-secreting cells in PBMCs correlated with those in LILs (rho=0.599, p=0.044 and rho=0.716, p=0.004, respectively). CONCLUSIONS: These data provide further evidence of the immunomodulatory role of the CD4(+)CD25(+) regulatory T lymphocytes in chronic HCV infection.

Dendritic cell maturation induced by delivery of ultraviolet-mediated apoptotic colorectal cancer cell lines.

BACKGROUND: Dendritic cells (DCs) play a critical role in the immune response. The aim of this study was to investigate apoptotic tumor cells as an antigen source for DC maturation. MATERIALS AND METHODS: We compared the efficacy of ultraviolet (UV)-irradiation with that of gamma-irradiation in the induction of apoptosis of tumor cells. Phenotypic and functional changes of DCs were analyzed after co-incubation with UV-irradiated tumor cells. RESULTS: UV-irradiation (1.8 J/cm2) was a more reliable method of inducing apoptosis than gamma-irradiation (20,000 rad). The expression of costimulatory molecules on DCs was upregulated after co-incubation with UV-irradiated tumor cells. When we performed allogeneic mixed lymphocyte reaction assay, UV-irradiated tumor cells-pulsed DCs stimulated allogeneic T lymphocytes more efficiently than DCs pulsed with gamma-irradiated cells (HT29, p = 0.0419 and WiDr, p = 0.0076). CONCLUSION: UV-irradiation reliably induces apoptosis in cultured colorectal cancer cells. DCs mature in function and expression of costimulatory molecules after co-incubation with apoptotic tumor cells. The clinical implications warrant further study.

Differential effect of cytotoxic T lymphocyte variant epitopes on generation and cytotoxicity in chronic hepatitis C virus infection.

Human leukocyte antigen (HLA) B44-restricted, hepatitis C virus (HCV)-specific cytotoxic T lymphocytes (CTLs) recognize HCV nucleoprotein amino acid residues 88-96 as an epitope. We previously reported the existence of variant peptide sequences at the epitope locus in three of 27 patients with HCV infection and HLA B44. Here we studied the effects of the variant peptide sequences on generation and cytotoxicity of CTLs. Two of the three variant peptides generated CTLs poorly although they activated well the cytotoxicity of CTLs. Such a differential activation of proliferation and cytotoxicity may contribute to the emergence of HCV with variant epitopes for CTLs.

Contribution of diuretic therapy with human serum albumin to the management of ascites in patients with advanced liver cirrhosis: A prospective cohort study.

The number of available studies on the role of human serum albumin (HSA) in the treatment of cirrhotic ascites is currently limited. In this study, we aimed to investigated the parameters associated with diuretic therapy with HSA in patients with advanced cirrhotic ascites. The patient inclusion criteria were cirrhotic ascites and a serum albumin (Alb) concentration of <3.5 g/dl. A total of 49 patients registered and 38 patients were ultimately included in this study. The enrolled patients were mainly treated with oral spironolactone and furosemide, which were not specified; the HSA amount was also not specified, although the administration period was set to a maximum of 7 days. Our results demonstrated that the administration of HSA significantly increased the serum levels of Alb [0.97 g/dl; two-sided 95% confidence interval (CI): 0.83-1.11 g/dl] and decreased body weight (-2.24 kg; 95% CI: -3.06 to -1.43 kg), hematocrit ratio (0.96; 95% CI: 0.94-0.98) and plasma renin concentration (day 4; geometric mean fold change, -0.1528; 95% CI: -0.2510 to -0.0545; log-transformed data) in patients with advanced cirrhotic ascites. The observed weight loss was found to be correlated with the total amount of HSA administered (P=0.0012), as indicated by the results of the multiple linear regression analysis. In conclusion, this study confirmed the efficacy of HSA in patients with advanced cirrhotic ascites.

Hepatitis C patients on maintenance hemodialysis show complex immune disturbances in the peripheral blood.

Hepatitis C virus (HCV) infection is prevalent in patients with maintenance hemodialysis (HD). Although HCV affects the survival rate, antiviral treatment for HD patients is limited. Since impaired innate immunity has been proposed both in hepatitis C and in HD, we compared the immunologic features in periphery between these patients and controls. Thirty subjects were divided into four groups on the basis of HD and HCV infection. In peripheral blood mononuclear cells, NK subsets and their activation status were analyzed by flow cytometry. Cytokine productions were measured both in the culture supernatant and at the single cell level. In HCV-infected HD patients, CD56(dim) NK subset was decreased (13.1±3.7%, p=0.015) but had upregulated CD69 expression (10.4±4.2%, p=0.032) compared to the other groups. LPS effectively induced neither interferon (IFN)-γ nor tumor necrosis factor (TNF)-α in NK cells of both HCV-infected and -uninfected HD patients, while TNF-α-producing monocytes were increased in HCV-infected HD patients as compared to the uninfected. These findings indicate that chronic HCV infection affects innate immunity independently of HD.

Interleukin-4 and CpG oligonucleotide therapy suppresses the outgrowth of tumors by activating tumor-specific Th1-type immune responses.

Because IL-4 and CpG oligodeoxynucleotides (CpG-ODNs) are immune stimulants, we evaluated the antitumor effects of IL-4 gene therapy and CpG-ODN treatment in a poorly immunogenic murine cancer model. We used a murine colorectal cancer MC38 cell line overexpressing the IL-4 gene (MC38-IL4). Incubation with MC38-IL4 and CpG-ODN enhanced bone marrow-derived dendritic cell (DC) maturation in vitro. In addition, interferon (IFN)-γ production was significantly increased in naïve splenocytes after they were coincubated with MC38-IL4 and CpG-ODN. When mice bearing MC38 wild-type tumors were inoculated subcutaneously with MC38-IL4 cells and CpG-ODN, the outgrowth of established parental tumors was significantly suppressed compared to those in the MC38-IL4-treated group (IL-4 vs. IL-4 + CpG-ODN, p=0.015). A marked infiltration of CD8+ cells in the established parental tumors of mice treated with MC38-IL4 and CpG-ODN was confirmed by immunohistochemical analyses (MC38-IL4, 2.8 ± 1.9 cells/field vs. MC38-IL4 + CpG-ODN, 20.7 ± 15.3 cells/field, p=0.027). Significant tumor-specific cytolysis was detected when splenocytes of MC38-IL4 + CpG-ODN-treated mice were stimulated by γ-irradiated MC38-IL4 cells and CpG-ODN twice weekly in vitro and used as effector cells in a chromium-release assay (32.2 ± 3.5% for MC38 cells vs. 3.2 ± 1.1% for YAC-1 cells; at an effector to target ratio of 40). These results suggest that IL-4 and CpG-ODN treatment promotes potent Th1-type antitumor immune responses. Therefore, the combination of IL-4 gene therapy and CpG-ODN treatment for cancer should be evaluated in clinical trials.