Ruslana Alper

Directed Differentiation of Human Embryonic Stem Cells into Functional Retinal Pigment Epithelium Cells

Dysfunction and loss of retinal pigment epithelium (RPE) leads to degeneration of photoreceptors in age-related macular degeneration and subtypes of retinitis pigmentosa. Human embryonic stem cells (hESCs) may serve as an unlimited source of RPE cells for transplantation in these blinding conditions. Here we show the directed differentiation of hESCs toward an RPE fate under defined culture conditions. We demonstrate that nicotinamide promotes the differentiation of hESCs to neural and subsequently to RPE fate. In the presence of nicotinamide, factors from the TGF-beta superfamily, which presumably pattern RPE development during embryogenesis, further direct RPE differentiation. The hESC-derived pigmented cells exhibit the morphology, marker expression, and function of authentic RPE and rescue retinal structure and function after transplantation to an animal model of retinal degeneration caused by RPE dysfunction. These results are an important step toward the future use of hESCs to replenish RPE in blinding diseases.

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

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.

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.

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.

Immunomodulation of experimental colitis: the role of NK1.1 liver lymphocytes and surrogate antigens--bystander effect.

The imbalance between Th1 pro‐inflammatory and Th2 anti‐inflammatory cytokine‐producing cells plays a major role in the pathogenesis of inflammatory bowel disease (IBD). Induction of oral tolerance to colitis‐extracted proteins was previously shown to down‐regulate the anti‐colon immune response, thereby alleviating experimental colitis. Immune bystander effect and liver‐associated lymphocytes expressing the NK1.1 marker (NK1.1+ LAL) have been suggested as being important in tolerance induction. The aims of the present study were to determine whether oral administration of inflammatory and non‐inflammatory colon‐extracted proteins of different species can induce peripheral immune tolerance and alleviate experimental colitis; and to examine the role of NK1.1+ LAL in oral tolerance induction. Colitis was induced in C57/B6 mice by intracolonic instillation of trinitrobenzene sulphonic acid (TNBS). Mice received six oral doses of colonic proteins extracted from TNBS‐colitis colonic wall, or normal colonic wall, from four different species. Standard clinical, macroscopic, and microscopic scores were used for colitis assessment. Serum interferon γ (IFNγ) and interleukin 10 (IL10) levels were measured by ELISA. To evaluate the role of NK1.1+ LAL in maintaining the balance between immunogenic and tolerogenic subsets of cells, their cytotoxicity functions were tested in tolerized and non‐tolerized‐mice. The administration of mouse‐derived colitis‐extracted proteins, or of surrogate proteins extracted from normal mouse colon, or from rat or human inflammatory colons, was found to alleviate experimental colitis. Tolerized mice had less diarrhoea; showed a marked reduction of colonic ulceration, intestinal and peritoneal adhesions, wall thickness, and oedema; and demonstrated a significant improvement of all microscopic parameters for colitis. Induction of tolerance led to an increase in IL10 and a decrease in IFNγ serum levels. NK1.1+ LAL cytotoxicity function increased markedly in tolerized mice. In contrast, mice fed with proteins extracted from normal rat, rabbit, and human colon, or from rabbit inflammatory colon, developed severe colitis, with a marked increase in IFNγ and a decrease in IL10 serum levels, and down‐regulation of NK1.1+ LAL function. This study has shown that oral tolerance can be induced in experimental colitis by means of the feeding of surrogate antigens; this alleviates experimental colitis. NK1.1+ LAL cytotoxicity function is associated with peripheral tolerance induction and may help to maintain the Th1/Th2 immune balance. Copyright

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‐γ (IFN‐γ) 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‐γ 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.

Suppression of Hepatocellular Carcinoma Growth via Oral Immune Regulation towards Tumor-Associated Antigens Is Associated with Increased NKT and CD8+ Lymphocytes

Background: Oral immune regulation towards viral proteins was previously shown to modulate the anti-HBV immune response. Adoptive transfer of orally immunomodulated lymphocytes suppressed the growth of hepatocellular carcinoma (HCC) expressing HBsAg in athymic mice. NKT lymphocytes play a role in the defense against tumor growth. Aim: To evaluate the effect of oral immune regulation towards HCC-associated antigens or HBV proteins on growth of HBsAg-expressing HCC, and to determine the role of NKT lymphocytes in immune modulation. Methods: Sublethally irradiated athymic Balb/c mice were injected with 107 human hepatoma cells followed 10 days later by transplantation of 2 × 106 splenocytes from naive donor mice. Immune modulation was performed via feeding of HCC-extracted proteins or HBV antigens (HBsAg + Pre S1 + Pre S2). The control group was fed with bovine serum albumin (BSA). Mice were followed for survival, tumor volume, and serum α-fetoprotein levels. To determine the role of NKT cells in tumor suppression, cytokine expression and FACS analysis for CD4+, CD8+, and NK1.1+ T lymphocyte subsets were performed. Results: Oral immune regulation towards HCC-extracted proteins induced complete tumor suppression in recipient mice. Mortality rates were 0% in HCC-immune-regulated mice, compared with an 80% mortality rate using HBV antigens and a 100% mortality rate in control mice. Oral immune regulation towards HCC prevented weight loss. No visible tumor mass was observed in orally immune-regulated mice as compared with 112 mm3 in controls. Serum αFP levels were 0.9, 378 and 1,358 ng/ml in HCC, HBV immune-regulated and controls, respectively. Immune regulation towards HCC antigens significantly increased the NK1.1+ T lymphocytes/CD4+ and CD8+/CD4+ ratios. IFNγ production increased two-fold. Conclusion: Oral immune regulation towards HCC antigens effectively enhanced the anti-tumor immune response, thus suppressing the growth of HCC in mice. This effect was associated with an increased NKT,CD8+/CD4+ lymphocyte ratio and may be mediated via enhancement of IFNγ production.