Elazar Rabbani

Adoptive transfer of regulatory NKT lymphocytes ameliorates non‐alcoholic steatohepatitis and glucose intolerance in ob/ob mice and is associated with intrahepatic CD8 trapping

The aim of this study was to determine the effect of adoptive transfer of regulatory natural killer T (NKT) lymphocytes on the metabolic disorder in leptin‐deficient ob/ob mice, which feature depletion and defective function of NKT and CD4 lymphocytes. Leptin‐deficient ob/ob mice were subjected to transplantation of 1 × 106 of either ob/ob or wild‐type‐derived NKT lymphocytes, or to transplantation of either ob/ob or wild‐type‐derived splenocytes. The effect on hepatic fat content was measured by magnetic resonance imaging (signal intensity index) and histology, using the steatohepatitis grading scale. The degree of glucose intolerance was measured by an oral glucose tolerance test (GTT). Adoptive transfer of wild‐type or ob/ob‐derived regulatory NKT cells led to a 12% decrease in hepatic fat content. A significant histological shift from macrosteatosis to microsteatosis was observed. Marked improvement in the GTT was noted in wild‐type or ob/ob‐derived NKT recipients. Metabolic effects were associated with a significant decrease in peripheral and intrahepatic CD4/CD8 lymphocyte ratios. Intrahepatic CD8 trapping was observed in all responders. Serum interleukin 10 levels decreased significantly. In conclusion, adoptive transfer of a relatively small number of regulatory NKT lymphocytes into ob/ob mice results in a significant reduction in hepatic fat content, a shift from macro to microsteatosis, and significant improvement in glucose intolerance. These effects were associated with decreased peripheral and intrahepatic CD4/CD8 ratios and decreased interleukin 10 levels. The results further support a role for regulatory NKT lymphocytes in the pathogenesis of non‐alcoholic steatohepatitis in the leptin‐deficient murine model. Copyright

Treatment of experimental colitis by oral tolerance induction: a central role for suppressor lymphocytes

Treatment of experimental colitis by oral tolerance induction: a central role for suppressor lymphocytes

Glucocerebroside Ameliorates the Metabolic Syndrome in OB/OB Mice

Glucocerebroside (GC) is a naturally occurring glycolipid that may alter natural killer T (NKT) cell function. To determine the effect of GC on the metabolic derangements and immune profile in leptin-deficient mice, Ob/Ob mice were treated by daily injections of GC for 8 weeks and followed for various metabolic and immunological parameters. Marked amelioration of the metabolic alterations characteristic of leptin-deficient mice was observed in GC-treated animals compared with controls. A significant decrease in liver size and hepatic fat content were observed in GC-treated mice. Near-normalization of glucose tolerance and decreased serum triglyceride levels were observed. Fluorescence-activated cell sorting analysis of peripheral and intrahepatic lymphocytes revealed a 1.6-fold increase of the peripheral/intrahepatic NKT lymphocyte ratio. A 33% decrease of serum interferon-γ level and a 2.6-fold increase of serum interleukin 10 level were noted in GC-treated mice. Immune modulation by GC may have a role in the treatment of nonalcoholic steatohepatitis and other immune-mediated disorders.

NKT and CD8 lymphocytes mediate suppression of hepatocellular carcinoma growth via tumor antigen-pulsed dendritic cells

Dendritic cells (DCs) are antigen presenting cells that play a role in T‐cell activation. Liver‐associated natural killer T lymphocytes (NKTs) are a unique subset of lymphocytes that may be important in antitumor immunity. Hepatitis B virus (HBV)‐associated hepatocellular carcinoma (HCC) expresses hepatitis B virus surface antigen (HBsAg) on its cell surface and may serve as a tumor‐associated antigen. The aim of the study was to evaluate the antitumor effect of DC pulsed with tumor or viral‐associated antigens in HBV‐expressing HCC in mice and to determine the role of NKT lymphocytes in this process. Balb/c mice were sublethally irradiated and transplanted with Hep3b HCC cell line, followed by transplantation of naive splenocytes. DCs were separated using CD11c beads and pulsed with HBV‐enveloped proteins (group A), HCC cell lysate (group B), or BSA (control group C). Mice were followed for survival and tumor size. To determine the mechanism of the antitumor effect, intrasplenic and intrahepatic lymphocyte subpopulations were analyzed by FACS for NKT, CD4 and CD8 markers. Tumor‐associated antigens‐specific IFNγ ELISPOT, T‐cell proliferation assays and serum cytokine analysis were performed. Treatment with tumor‐associated antigen‐pulsed DC significantly improved survival (40% and 50% as compared with 0% in groups A, B, and control group C, respectively; p < 0.005). Tumor size decreased to 12.8 ± 0.4 and 0 from 60.4 ± 0.9 mm3 in groups A, B, and control group C, respectively (p < 0.005). Adoptive transfer of HBV or Hep3b‐associated antigens‐pulsed DC induced a 6‐fold increase in peripheral CD8+ lymphocytes (from 1% in control mice to 6% and 5.5% in groups A and B, respectively), along with a decrease in CD4+ lymphocytes (from 3.5% in controls to 1.4% and 2.3% in A and B, respectively; p < 0.005). The CD8+/CD4+ ratio increased from 0.28 in controls to 4.28 and 2.39 in groups A and B, respectively (p < 0.005). Intrasplenic NKT cells increased from 7% in control mice to 7.98% and 14.6% in groups A and B, respectively. In contrast, an opposite shift was observed inside the liver. Intrahepatic lymphocyte analysis showed a marked increase in CD4+ and a decrease in CD8+ lymphocytes in treated groups. The intrahepatic CD4+ number increased from 0.5% in controls to 2.15% and 25.8% in groups A and B, respectively (p < 0.005). In contrast, a significant decrease in the intrahepatic CD8+ numbers was observed (from 7% in controls to 1.0% and 2.4% in groups A and B, respectively; p < 0.005). A significant increase was noted in HBV‐specific IFNγ spot‐forming T‐cell colonies from 0.0 to 8.8 ± 1.7 and 1.8 ± 2.9 in groups C, A, and B, respectively (p < 0.005). Similarly, a significant increase in the HBV‐specific T‐cell stimulation index, from 0.8 ± 0.2 to 7.2 ± 0.4, in groups C and B, respectively, was noted (p < 0.002). IFNγ and IL12 serum levels increased significantly in treated groups. IFNγ and IL12 serum levels increased to 380 ± 30 and 400 ± 20, and 960 ± 40 and 760 ± 60 in groups A and B, compared with 150 ± 16 and 490 ± 40 pg/ml in control mice (p < 0.005). Tumor antigen‐pulsed DCs effectively suppressed the growth of hepatocellular carcinoma in mice. This effect was associated with enhanced NKT and CD8+ lymphocyte function and augmentation of the antitumor/antiviral‐specific IFNγ production.

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, IFNγ, 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 IFNγ positive T cell clones (62.9%) was noted. In addition, a decrease in the IL10 γ 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.

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 Crohns 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, IFNγ 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 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 × 06 NKT cells per mouse). Group D mice did not undergo NKT cell transplantation. Group E mice were transplanted with 1 × 106 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 IFNγ, 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 IFNγ and IL12, and of IL4. Ex‐vivo modulation of NKT lymphocytes holds promise as a novel mode of immune therapy for HCC.

Amelioration of non-alcoholic steatohepatitis and glucose intolerance in ob/ob mice by oral immune regulation towards liver-extracted proteins is associated with elevated intrahepatic NKT lymphocytes and serum IL-10 levels.

Non‐alcoholic steatohepatitis (NASH) is a common cause of cryptogenic cirrhosis in the Western world. In an animal model of NASH, leptin‐deficient ob/ob mice present with alterations in number and function of hepatic NKT and peripheral CD4 lymphocytes. Oral immune regulation is a method to alter the immune response towards orally administered antigens. To determine the effect of oral immune regulation towards liver‐extracted proteins on the metabolic disorders in ob/ob mice, ob/ob mice and their lean littermates were orally administered liver extracts from wild‐type or ob/ob mice or bovine serum albumin for 1 month. The effect of treatment on hepatic fat content was measured by magnetic resonance imaging (MRI) and using a histological steatohepatitis grading scale. Glucose tolerance was measured by an oral glucose tolerance test (GTT). T lymphocyte subpopulations were assessed by flow cytometry analysis. Induction of immune regulation by oral presentation of liver‐extracted proteins resulted in a significant 18% reduction of the hepatic fat content in ob/ob mice fed with either wild‐type or ob/ob liver extracts for 1 month. The MRI signal intensity index in treated mice decreased to 0.48 and 0.51, respectively, compared with 0.62 in BSA‐fed controls (p = 0.037 and p = 0.019, respectively), while the histological steatohepatitis score decreased in both treated groups to 2.0, compared with 2.4 in BSA‐fed controls (p = 0.05). A significant improvement in GTT was noted in treated ob/ob mice. These changes were accompanied by a marked increase in the intrahepatic NKT lymphocyte population in mice fed with proteins extracted from both wild‐type and ob/ob mice (46.96% and 56.72%, respectively, compared with 26.21% in BSA‐fed controls; p < 0.05) and a significant elevation in serum IL‐10 levels. Oral immune regulation towards liver extracted proteins in leptin‐deficient mice resulted in a marked reduction in hepatic fat content and improved glucose tolerance. This effect was associated with a significant increase in the intrahepatic NKT lymphocyte population and serum IL‐10 levels, suggesting a Th1 to Th2 immune shift. Immune regulation towards disease‐associated antigens holds promise as a new mode of therapy for NASH. Copyright

Alleviation of acute and chronic graft-versus-host disease in a murine model is associated with glucocerebroside-enhanced natural killer T lymphocyte plasticity.

Background. Natural killer T (NKT) lymphocytes play a role in graft-versus-host disease GVHD (GVHD). Glucocerebroside (GC) is a naturally occurring glycolipid that plays a role in the immune modulation of NKT lymphocytes. This study aims to determine the effect of GC in murine models of semiallogeneic/acute and chronic GVHD. Methods. Acute and chronic GVHD were generated by fusion of splenocytes from C57BL/6 to (C57BL/6xBalb/c) F1 mice, and from B10.D2 donor mice into Balb/c, respectively. Recipients were treated daily with GC. Histological and immunological parameters of GVHD were assessed. Results. Treatment with GC significantly alleviated GVHD in both models The beneficial effect of GC was associated with an increase in the intrahepatic/peripheral NKT lymphocyte ratio in the semiallogeneic/acute model. This ratio was decreased in the chronic GVHD model. A significant increase in intrahepatic CD8+ lymphocyte trapping was noted in the semiallogeneic/acute model, whereas the opposite effect was observed in the chronic GVHD model. Administration of GC led to decreased serum interferon-&ggr; and increased serum interleukin-4 levels in the Th1-mediated model, whereas the opposite effect was observed in the Th2-mediated models. Conclusions. GC ameliorates GVHD in both Th1- and Th2-mediated murine models, suggesting it is able to differentially affect the immune system. GC may alleviate immunologically incongruous disorders, and may be associated with “fine tuning” of immune responses.

Induction of oral tolerance towards hepatitis B envelope antigens in a murine model

BACKGROUND Hepatitis B virus (HBV) is a non-cytopathic virus, and the hepatocellular injury that occurs as a consequence of HBV infection is mediated by the host antiviral immune response. Subjects with natural tolerance to HBV have minimal or no liver injury despite chronic viremia. We have shown that immune tolerance towards viruses can be induced by oral administration of viral proteins. AIMS To test whether oral induction of tolerance can be induced towards HBV antigens, and whether oral tolerance induction downregulates preexisting anti-HBV immune response. METHODS Oral tolerance was induced via feeding of five low oral doses of HBV proteins (HBsAg+preS1+preS2, BioHepB). This was followed by two inoculations with the BioHepB vaccine. Humoral immune tolerance was evaluated by measuring serum levels of anti-HBs antibody titers at monthly intervals. To determine if oral tolerance induction downregulates pre-existing anti-HBs immunity, mice were inoculated twice with the BioHepB vaccine, followed by feeding of BioHepB-HBV proteins. RESULTS Feeding of HBV proteins markedly inhibited production of anti-HBs antibodies in naive mice. Anti-HBs titers were 45 versus 135 mIU/ml, in tolerized versus non-tolerized controls (P<0.005). Moreover, oral tolerance induction effectively down-regulated pre-existing immunity and reduced the anti-HBs titers in previously immunized mice to 112 versus 223 mIU/ml, in tolerized compared with non-tolerized controls (P<0.01). CONCLUSIONS Induction of oral tolerance towards HBV proteins downregulates the antiviral humoral immune response in naive mice, and in the presence of preexisting anti-HBV immunity. This approach should be further investigated as a method for alleviation of antiviral-mediated liver injury in chronic HBV hepatitis.