Oral mixture of autologous colon-extracted proteins for the Crohn's disease: A double-blind trial.

AIM: To evaluate the safety and efficacy of oral administration of Alequel™, an autologous protein-containing colon extract. METHODS: A total of 43 patients were enrolled in a randomized, placebo-controlled, double-blind trial. Patients were orally administered with autologous protein-containing colon extract three doses of autologous study drug per week for 15 wk, for a total of 45 doses. Patients were followed for safety parameters. Remission was defined as a Crohn's disease activity index (CDAI) score of less than or equal to 150. All patients were followed for changes in subsets of T cells by fluorescence-activated cell sorting analysis. RESULTS: Analysis was performed on a total number of evaluable patients of 14 in the study drug group and 15 in the placebo group. Treatment was well tolerated by all patients. No major treatment-related adverse events were reported or observed in any of the treated patients during the feeding or follow-up periods. Between weeks 6 and 9 of the study, six of the 14 (43%) evaluable subjects who received the study drug achieved a CDAI of 150 or lower. In contrast, five of the 15 (33%) evaluable subjects in the placebo group achieved remission. Between weeks 9 and 12, the remission rates were 50% and 33% for the drug group and placebo group, respectively. Among the drug-treated subjects who achieved remission, the effect of the drug was judged as stable in eight of the 14 subjects as measured by at least two CDAI scores indicating remission in the 15-wk treatment period. A decreased percentage of peripheral natural killer T regulatory cells (a decrease of 28% vs an increase of 16%) and an increased ratio of CD4(+)/CD8(+) T lymphocytes (an increase of 11% vs a decrease of 9%) were noted in subjects with a significant clinical response. CONCLUSION: Oral administration of the autologous colonic extract could be a safe and effective for the treatment of patients with moderate to severe Crohn's disease.

Tracing hepatitis B virus to the 16th century in a Korean mummy.

UNLABELLED: A rare find of a mummified child from the 16th century AD, in Korea, with relatively preserved organs, enabled a search for ancient hepatitis B virus (aHBV) DNA sequences from laparoscopic-derived liver biopsies. Analysis of the complete aHBV genome (3,215 base pairs) revealed a unique HBV genotype C2 (HBV/C2) sequence commonly spread in Southeast Asia, which probably represents an HBV that infected the Joseon Dynasty population in Korea. Comparison of the aHBV sequences with contemporary HBV/C2 DNA sequences revealed distinctive differences along four open reading frames. Genetic diversity between contemporary and recovered aHBV/C2 DNA may be the result of immunologic, environmental, and/or pharmacologic pressures. The calculated time of most recent common ancestor suggests that the Korean HBV sequence origin dates back at least 3,000 years and possibly as long as 100,000 years. This isolate most likely represents the earliest human HBV sequence that colonized Southeast Asia by human migration. CONCLUSION: This study describes the complete sequence of the oldest HBV isolate and the most ancient full viral genome known so far.

Oral administration of OKT3 monoclonal antibody to human subjects induces a dose-dependent immunologic effect in T cells and dendritic cells.

INTRODUCTION: Parenteral OKT3 is used to treat transplant rejection and a humanized anti-CD3 Mab has shown positive clinical effects in new onset diabetes. Oral administration of anti-CD3 has not been tested in humans, but suppresses autoimmunity in animal models. Beta-glucosylceramide enhances NKT cell and regulatory T cell activity and enhances the effects of oral anti-CD3 in animals. MATERIALS AND METHODS: Fifteen healthy volunteers (three per group) received orally administered OKT3 over a dose range of 0.2 to 5.0 mg daily with or without beta-glucosylceramide 7.5 mg for 5 days. Safety and immune parameters were measured on days 5, 10, and 30. RESULTS AND DISCUSSION: Oral OKT3 enhanced T cell proliferation, suppressed Th1 and Th17 responses by 43% and 41%, respectively, increased TGF-beta/IL-10 expression and decreased IL-23/IL-6 expression by dendritic cells, and affected the IgG repertoire as measured by antigen arrays. Co-administration of oral beta-glucosylceramide induced similar effects. No side effects were observed and no subjects developed human anti-mouse antibodies. CONCLUSION: These findings demonstrate that oral anti-CD3 monoclonal antibody is safe and biologically active in humans and presents a new avenue for the treatment of autoimmune diseases.

A double-blind clinical trial for treatment of Crohn's disease by oral administration of Alequel, a mixture of autologous colon-extracted proteins: a patient-tailored approach.

OBJECTIVES: In this study, we evaluated the safety and efficacy of a personalized mode of treatment for Crohn's disease (CD) by oral administration of Alequel an extract of autologous colonic proteins. METHODS: Thirty-one patients with moderate to severe CD were enrolled in a 27-wk randomized, double-blind, placebo-controlled trial. Patients were randomized to receive either a placebo or the study drug prepared from autologous colonic extract. RESULTS: Oral administration of autologous colonic proteins resulted in clinical remission (58% vs 29%; 46.6% vs 26.6%, using an intention to treat analysis, p= NS), clinical response (67% vs 43%; 53.3% vs 40%, using an intention to treat analysis, p= NS) and improved quality of life (Inflammatory Bowel Disease Questionnaire score improvement 43%vs 12%) in the drug study group, compared to placebo group. No treatment-related adverse events were noted. Only in the study-drug-treated cohort who achieved clinical remission (DR), there was a decreased number of subject-specific, antigen-directed, IFNgamma spot-forming colonies. DR subjects had a lower initial C-reactive protein level than DNOR or placebo subjects, an increased percentage of peripheral blood nature killer T cells, and an increased CD4+/CD8+ T-cell ratio throughout the period of drug administration. CONCLUSIONS: Oral administration of Alequel is a safe method for treatment of patients with moderate to severe CD, and its efficacy needs to be proven. Several markers may be applicable as surrogate markers for the clinical effect.

Suppression of hepatocellular carcinoma growth in mice via leptin, is associated with inhibition of tumor cell growth and natural killer cell activation.

BACKGROUND/AIMS: Leptin exerts potent immune modulatory properties. The aim of this study was to determine leptin's anti-tumor effect in a murine model of hepatocellular carcinoma (HCC). METHODS: In vivo, athymic nude mice were transplanted with Hep3B cells, followed by daily leptin administration for 6 weeks. RESULTS: Leptin administration induced a significant reduction in tumor size, improved survival rate, and was associated with a significant increase in peripheral natural killer (NK) cell number. Splenocytes from leptin-treated mice featured decreased expression of CIS mRNA. SCID mice featured a similar leptin-associated tumor suppression. In contrast, NK-deficient SCID-beige mice developed larger tumors which were unresponsive to leptin. NK cells incubated in vitro with increasing doses of leptin demonstrated increased cytotoxicity and proliferation. Incubation of leptin with hepatoma cells induced a dose-dependent reduction in proliferation, suggesting a direct anti-tumor effect. Leptin induced increased mRNA expression of STAT2 and SOCS1 on HCC cell lines. CONCLUSIONS: Leptin administration induces a significant suppression of human HCC. This effect is mediated by induction of natural killer cell proliferation and activation, along with direct inhibition of tumor growth. Decreased NK expression of inhibitory CIS and over-expression of the antiproliferative STAT2 and SOCS1 proteins in HCC lines may underline the anti-cancerous effects of leptin.

Glucocerebroside treatment ameliorates ConA hepatitis by inhibition of NKT lymphocytes.

Concanavalin A (ConA) induces natural killer T (NKT) cell-mediated liver damage. Glucocerebroside (GC) is a naturally occurring glycolipid. Our aims were to determine the effect of GC in a murine model of ConA-induced hepatitis. Mice in groups A and B were treated with GC 2 h before and 2 h following administration of ConA, respectively; group C mice were treated with ConA; group D mice was treated with GC; group E mice did not receive any treatment. Liver damage was evaluated by serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels and liver histology. The immune effect of GC was determined by fluorescence-activated cell sorter analysis of intrahepatic and intrasplenic NKT lymphocytes, measurement of cytokine levels, and Western blot analysis for STAT 1, 4, 6, and NF-kappaB expression. The effect of GC on NKT cell proliferation was assessed in vitro. Serum AST and ALT levels were markedly reduced in GC-treated group A mice compared with nontreated group C animals, and histological damage was markedly attenuated in group A. The beneficial effect of GC was associated with a 20% decrease of intrahepatic NKT lymphocytes, significant lowering of serum IFN-gamma levels, and decreased STAT1 and STAT6 expression. In vitro administration of GC led to a 42% decrease of NKT cell proliferation in the presence of dendritic cells but not in their absence. Intraperitoneally administered radioactive GC was detected in the liver and bowel. Administration of GC led to amelioration of ConA hepatitis associated with an inhibitory effect on NKT lymphocytes. GC holds promise as a new immune-modulatory agent.

Oral immune regulation using colitis extracted proteins for treatment of Crohn's disease: results of a phase I clinical trial.

AIM: To evaluate safety and possible efficacy of induction of oral immune regulation using colitis extracted proteins (CEP) in Crohn's disease (CD) subjects. METHODS: Ten CDs were treated orally with autologous CEP thrice weekly for 16 wk. Subjects were monitored for CDAI and IBDQ. Immune modulatory effect was assessed by T-lymphocyte FACS analysis, CEP-specific IFNgamma ELISPOT assay and cytokine levels. RESULTS: Induction of oral immune regulation significantly ameliorated disease activity. All (10/10) subjects had clinical response (CDAI < or = 70) and 7/10 achieved clinical remission (CDAI < or = 150). Significant increase in mean IBDQ score was noted (134+/-9 vs 164+/-12). No treatment-related adverse events were noted. High levels of CEP-specific IFNgamma spot forming colonies were detected in five subjects prior to treatment and in all five, a marked decrease was observed. The CD4+/CD8+ lymphocyte ratio and peripheral NKT cell numbers increased significantly, in 7/10 and in 5/10 subjects, respectively. Significant increase in serum IL-10 and IL-4 levels was observed in 7/10 subjects during treatment period. CONCLUSION: Immune regulation via oral administration of CEP is a safe and possibly effective treatment for subjects with moderate CD and may provide means of antigen-specific immune modulation.

Suppression of hepatocellular carcinoma by transplantation of ex-vivo immune-modulated NKT lymphocytes.

NKT cells are a regulatory subset of T lymphocytes with immune modulatory effects and an important role in anti-tumor immunity. The feasibility of "ex-vivo education" of NKT cells has recently been demonstrated. To evaluate the anti-tumor effect of ex-vivo immune-modulated NKT lymphocytes in a murine model of hepatocellular carcinoma. Athymic Balb/C mice were sublethally irradiated and transplanted with human Hep3B HCC. NKT cells prepared from immunocompetent Balb/C mice were pulsed ex vivo with HCC-derived antigens (Group A), Hep3B cells (group B) or BSA (group C), and adoptively transferred into HCC harboring mice (1 x 0(6) NKT cells per mouse). Group D mice did not undergo NKT cell transplantation. Group E mice were transplanted with 1 x 10(6) NKT cells from HBV-immunized donors. Mice were followed for tumor size and weight. To determine the mechanism of the anti-tumor effect, intrasplenic lymphocyte populations were analyzed by FACS for NKT, CD4+ and CD8+ lymphocyte subpopulations; STAT 1, 4 and 6 expression in splenocytes was assessed by Western blot, and serum cytokine levels were measured by ELISA. Adoptive transfer of NKT cells pulsed with HCC-derived antigens (group A) and NKT cells from immunized donors (group E) resulted in complete disappearance of tumors within 4 weeks and attenuated weight loss (6.5% and 7% in groups A and E, respectively). In contrast, mice in groups B, C, and D developed large, necrotic tumors and severe weight loss (21%, 17% and 23% weight loss in groups B, C, and D, respectively). NKT/CD4 and CD8/CD4 ratios were significantly increased in groups A and E (12.3 and 17.6 in groups A and D, respectively, compared to 6.4, 4.8 and 5.6 in groups B, C and D, respectively, for the NKT/CD4 ratio; 41 and 19.8 in groups A and E, respectively, compared to 6.5, 11.8 and 3.2 in groups B, C, and D, respectively, for the CD8/CD4 ratio). Expression of the transcription factor STAT4 was evident in group A, but not in groups B-D. Serum IFNgamma, IL12 and IL4 levels were increased in groups A and E. Adoptive transfer of NKT lymphocytes exposed ex vivo by HCC-derived antigens loaded on dendritic cells and NKT cells from immunized donors led to suppression of HCC in mice. NKT-mediated anti-tumor activity was associated increased NKT and CD8+ T lymphocyte numbers, increased expression of STAT4, a marker for IL-12 activity and elevated serum levels of the proinflammatory cytokines IFNgamma and IL12, and of IL4. Ex-vivo modulation of NKT lymphocytes holds promise as a novel mode of immune therapy for HCC.

Induction of oral immune regulation towards liver-extracted proteins for treatment of chronic HBV and HCV hepatitis: results of a phase I clinical trial.

BACKGROUND: Anti-viral immunity can be modulated via oral feeding of viral proteins. Hepatitis B and C viral (HBV, HCV)-associated hepatocellular injury is mediated by a defective host anti-viral immune response. AIMS: To determine the effect of oral administration of a mixture of liver-extracted proteins with HBV/HCV proteins, on viral load, liver injury, and the anti-viral T-cell response of chronic HBV/HCV patients. METHODS: Fourteen patients with chronic HBV and 15 patients with chronic HCV were treated orally with hepatocyte-extracted proteins and HBV or HCV viral proteins for 24 weeks, and followed for an additional 26 weeks. Patients were monitored for HBV-DNA or HCV-RNA levels, liver enzymes and liver histology. Viral-directed T-cell immunity was assessed by IFNgamma and IL10 ELISPOT, viral-specific T-cell proliferation, cytotoxicity, and cytokines assays, and followed for peripheral natural killer T-cell (NKT) number. RESULTS: In both chronic HBV and HCV patients, oral administration of a mixture of selected liver-extracted proteins and viral proteins induced a favorable increase in viral-specific T-cell proliferation, and IFNgamma-secreting clones, along with a significant decrease in the anti-viral IL10-secreting T-cell clones. However, the effects of modulation of the anti-viral immunity differed between the HBV and HCV patients. In both groups, no major adverse events were noted. In chronic HBV patients, a significant decrease in viral load was observed in 5/14 (35.7%) of patients. HB surface antigen/HB nucleocapsid antigen scores on liver biopsy improved in 46.1% and 50%, respectively, and the histological necroinflammatory score improved in 4/13 (30.7%). Forty percent of the patients with elevated liver enzymes showed a favorable biochemical response. In contrast, an improvement in the histological necroinflammatory score was observed in only 2/12 (17%) of the chronic HCV patients. No significant decrease in HCV RNA was noted in any of these patients. CONCLUSIONS: Immune regulation of the anti-HBV/HCV immune response via oral administration of a mixture of liver-extracted and viral proteins significantly altered the viral-specific immunity. This effect was associated with clinical and virological improvements in chronic HBV patients.

Genotype prevalence, viral load and outcome of hepatitis B virus precore mutant infection in stable patients and in patients after liver transplantation.

OBJECTIVE: The precore mutant is detectable in most Israeli patients with persistent hepatitis B virus (HBV) infection. The aim of this study was to determine the prevalence of HBV genotypes, viral load and outcome of precore mutant infection in stable patients and in patients after liver transplantation. METHODS: The prevalence of HBV genotype and viral load were investigated in 81 patients with HBV precore mutant infection. Of these, 50 patients (40 males, 10 females; mean age 43.4 +/- 11.0 yr) underwent liver transplantation and were serum HBV DNA-negative by hybridization at the time of transplantation. Patients received long-term HBV immunoprophylaxis and immunosuppression, and lamivudine in cases of graft HBV recurrence. The remaining 31 patients were stable, with serum anti-HBe-positivity. Genotypes were tested by restriction fragment length polymorphism of an S gene amplicon. Precore mutations were studied with an INNO-LiPA probe assay. RESULTS: Follow-up was 46.6 +/- 37.7 months. Most of the transplanted group was of Middle Eastern origin (53.6%); the remainder were from Eastern Europe (21.4%), Western Europe and the USA (10.8%), Africa (7.1%), and Asia (7.1%). In the transplanted group, the pre-transplant HBV genotype D was the most prevalent (96%), while genotype A was found in only 4%. Eleven patients (22%) developed recurrent HBV infection post-transplantation. There were no differences in genotype distribution between patients with graft reinfection or lamivudine resistance and patients without recurrence. Mean viral load at recurrence was 148.4 x 10(6) +/- 60.4 x 10(6) copies/mL. The stable group had a similar origin and HBV genotype prevalence, but a lower mean viral load of 12.4 x 10(6) +/- 29.4 x 10(6) copies/mL (p = 0.007). The prevalence of mutations at the precore region and codon 28 was similar in both groups. CONCLUSIONS: The chronic precore mutant HBV-infected patients were characterized as follows: (i) genotype D was the most frequent genotype, (ii) the HBV genotype distribution was similar in patients with stable infection and after liver transplantation, (iii) viral load at recurrence was significantly higher than in stable infection, and (iv) HBV genotype was unrelated to the development of recurrence or lamivudine resistance in the tested population.

Adoptive transfer of ex vivo immune-programmed NKT lymphocytes alleviates immune-mediated colitis.

T lymphocyte-expressing natural killer (NK) cell markers (NKT cells) play a role in immune regulation. Our aim was to evaluate the in vivo effect of adoptive transfer of immune-programmed NKT cells. Colitis was induced in C57/B6 mice by 2,4,6-trinitrobenzenesulfonic acid. NKT, CD4, CD8 lymphocytes, and dendritic cells (DC) were prepared from spleens of naive mice, animals with colitis, and animals with colitis that were orally tolerized. Subsets of splenocytes, NKT, CD4, and CD8 and NKT+CD4, NKT+CD8, and NKT+DC lymphocytes were prepared. Assessment of the T helper cell type 1 (Th1)/Th2 cytokine secretion paradigm in vitro was performed before and following exposure to the antigen. Adoptive transfer of ex vivo immune-programmed lymphocytes from each group was performed into recipient mice, followed by colitis induction. Ex vivo exposure of NKT cells harvested from mice with colitis-to-colitis proteins [colitis-extracted proteins (CEP)] led to a Th2 cytokine shift. The interleukin (IL)-4/interferon-gamma (IFN-gamma) ratio increased for NKT harvested from colitis-harboring mice following exposure to CEP. Adoptive transfer of NKT lymphocytes harvested from colitis-harboring mice, which were ex vivo-educated, significantly alleviated experimental colitis in vivo. Intrahepatic NKT lymphocytes increased significantly in mice transplanted with NKT lymphocytes harvested from colitis-harboring donor mice, which were ex vivo-exposed to CEP, similar to mice transplanted with NKT lymphocytes harvested from tolerized donors. Exposure of NKT cells to the disease-target antigen induced a significant increase in the IL-4/IFN-gamma cytokine ratio. Adoptive transfer of a relatively small number of immune-programmed NKT cells induced a systemic Th1 to Th2-immune shift and alleviated immune-mediated colitis.

Treatment of chronic hepatitis B virus infection via oral immune regulation toward hepatitis B virus proteins.

OBJECTIVES: Hepatitis B virus (HBV) is a noncytopathic virus, and hepatocellular injury is mediated by a defective host antiviral immune response. We have previously shown that antiviral immunity can be modulated through oral feeding of viral proteins. The aims of this study were to determine the safety and efficacy of treatment of patients with chronic HBV by means of p.o. administration of HBV envelope proteins. METHODS: A total of 42 chronic HBV patients were treated p.o. with HBV envelope proteins (HBsAg+preS1+preS2), three times/wk for 20-30 wk, and followed for an additional 20 wk. Patients were monitored for HBV-DNA levels, liver enzymes, and liver histology. HBV-directed T cell immune modulation was assessed in vitro by HBV specific T cell-proliferation, cytotoxicity, IFN gamma, and IL10 ELISPOT assays, and reverse transcription-polymerase chain reaction cytokines assay. RESULTS: Favorable response in one of the primary endpoints was achieved in 28/42 patients (66.6%) by means of p.o. immune regulation. A significant decrease in viral load was observed in 15 patients (35.7%). HBsAg/HBcAg biopsy scores improved in 41% and 57.1% of patients, respectively. Histological improvement in liver necroinflammatory score was noted in 12/40 patients (30%). In all, 80% showed biochemical response. Five of 19 HBeAg positive patients (26.3%) became negative for HBeAg. A favorable augmentation in anti-HBV specific T cell response, with increased HbsAg specific T cell proliferation (78%), cytotoxicity (75%), and IFN gamma positive T cell clones (62.9%) was noted. In addition, a decrease in the IL10 gamma positive T cell clones was achieved (48.1%). Natural killer T (NKT) lymphocytes increased significantly in all treated patients. CONCLUSIONS: Immune regulation of the anti-HBV immune response via p.o. administration of HBV envelope proteins alleviated the immune-mediated liver injury while augmenting the effective antiviral immunity.

Early expression of interferon gamma following oral antigen administration is associated with peripheral tolerance induction.

Oral tolerance is the induction of immune hyporesponsiveness to orally administered antigens. It was described in association with a decrease in interferon gamma (IFNgamma) production by activated T cells. To determine the role of IFNgamma and IL10 in immunemodulation via oral tolerization. Colitis was induced by intracolonic instillation of trinitrobenzene sulfonic acid. Treated mice received five oral doses of colitis-extracted proteins (CEPs) every other day, starting immediately after colitis induction. Control mice received similar doses of bovine serum albumin. Colitis was assessed in both groups by standard clinical, micro- and macroscopic scores. IFNgamma and IL10 expression in splenic lymphocytes from both groups was tested by RT-PCR immediately after oral feeding, 1, 4, 8, 12, and 24 h thereafter and then every 24 h for 2 weeks. Feeding of CEPs markedly ameliorated experimental colitis. These mice gained weight and showed markedly improved macro- and microscopic parameters of colitis. Tolerized mice exhibited IFNgamma expression in splenic lymphocytes starting immediately following oral CEP immunization and up to 14 d thereafter. IL10 was expressed starting 1 h after CEP feeding and during the first 48 h thereafter. In contrast, non-tolerized control mice manifested IFNgamma expression starting on day 6 and had no IL10 expression. Early induction of IFNgamma expression by oral antigen may be associated with systemic tolerance in the experimental colitis setting. In contrast, late expression of IFNgamma is associated with a pro-inflammatory response in non-tolerized controls.

Genotypic and phenotypic resistance: longitudinal and sequential analysis of hepatitis B virus polymerase mutations in patients with lamivudine resistance after liver transplantation.

OBJECTIVE: Lamivudine-resistant strains appear in 27-62.5% of liver transplant recipients treated with lamivudine for hepatitis B virus (HBV) recurrence, and may lead to failure of antiviral therapy. In an extension of our previous study, we investigated the molecular events associated with the emergence of lamivudine-resistant mutants in this population. METHODS: Sequential serum samples from 10 consecutive patients with lamivudine resistance after liver transplantation were analyzed for viral genotype, precore mutants, and viral polymerase gene mutants (L528M, M552V, M552I) using restriction fragment length polymorphism. Quantitative analysis of HBV DNA was performed using hybridization assay and polymerase chain reaction. RESULTS: Eight patients (80%) were infected with genotype D and two (20%) with genotype C. Polymerase mutants (genotypic resistance) were identified in all the patients. Phenotypic resistance (rise in serum HBV DNA titers above the detection limit of the hybridization assay) developed in five patients (50%); of the remainder, three (30%) did not have phenotypic resistance, and two were primary nonresponders. Genotypic resistance was detected earlier than phenotypic resistance (median 285 days [range 42-510] vs median 387 days [range 320-420], p = 0.055). In five patients (50%), the emergence of the YMDD mutants took over the wild type; in three (30%), the YMDD mutant took over the wild type, but the wild type re-emerged during lamivudine therapy; and in two (20%), the YMDD mutants were detected in a mixture with the wild type (in different percentages). The mean pretreatment serum ALT level was significantly lower in the patients who did not develop phenotypic resistance (p = 0.0002). The M552I pure viral population was found mainly in these patients, and all retained stable graft function (median follow-up 33 months). A high pretreatment HBV DNA level (>50 x 10(6) copies/ml) was highly statistically significantly correlated with the rapid occurrence of phenotypic resistance (r = -0.90, p = 0.04). CONCLUSIONS: We reached the following conclusions: 1) In our area, liver transplant recipients who develop resistance to lamivudine given for recurrent HBV infection seem to be mainly infected with genotype D. 2) Re-emergence of the wild type can occur during lamivudine therapy. 3) Genotypic resistance precedes phenotypic resistance, although phenotypic resistance does not always follow genotypic resistance. 4) Quantitative determination of viremia and analysis of polymerase gene mutants are recommended for monitoring antiviral therapy of liver transplant patients with HBV reinfection in the graft.

Inducing oral immune regulation of hepatitis B virus envelope proteins suppresses the growth of hepatocellular carcinoma in mice.

BACKGROUND: Hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) expresses hepatitis B surface antigen (HBsAg) on its cell surface, and this may serve as a tumor-associated antigen. It was shown previously that adoptive transfer of immunity against HBsAg facilitates the suppression of experimental human HCC-expressing HBsAg in athymic mice. The authors recently showed that it was possible to augment the anti-HBV immune response through induction of oral immune regulation for HBV-associated antigens. The objective of this study was to evaluate the effect of oral immune regulation for HBV antigens on the growth of HBsAg-expressing HCC. METHODS: Recipient athymic Balb/c mice were irradiated sublethally and injected with 10(7) human hepatoma cells followed by the adoptive transfer of 2 x 10(6) splenocytes from donor mice. Four groups of donor Balb/c mice were studied: Two groups were immune modulated through oral administration of HBV antigens (HBsAg, PreS1, and Pre S2) or bovine serum albumin (BSA). Two control groups were immunized for HBsAg and fed HBV antigens or BSA. Recipient mice were followed for tumor volume and serum alpha-fetoprotein (aFP) levels. The humoral immune response was determined by measuring serum HBs antibodies. HBV specific T-cell immune modulation was assessed in vitro by HBV specific T-cell proliferation and interferon gamma (IFNgamma) ELISPOT assays as well as cytokine expression by reverse transcriptase-polymerse chain reaction assays. RESULTS: The adoptive transfer of orally immune modulated HBV splenocytes induced complete tumor suppression in recipient mice compared with control mice transplanted with nonimmune modulated cells (BSA), which showed significant tumor growth (serum aFP levels were 3.5 ng/mL and 2320.0 ng/mL, respectively). Control mice transplanted with anti-HBs immunized cells (with or without oral immune modulation) manifested similar tumor suppression (3.5 ng/mL and 0.5 ng/mL, respectively). Immunoregulation for HBV antigens augmented the HBV specific T-cell immune response, as manifested by an increase in HBV specific T-cell proliferation and IFNgamma ELISPOT assays in mice orally immune regulated with HBV proteins compared with naïve mice. Tumor suppression was mediated through increased IFNgamma production in immune regulated and immunized mice. CONCLUSIONS: The induction of oral immune regulation for HBV antigens modulated the antitumor immune response, thus suppressing the growth of HCC in mice. This effect was mediated by the enhancement of anti-HBV specific T-cell immunity.