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.

Background: b-Glucosylceramide, a naturally occurring glycolipid, exerts modulatory effects on natural killer T (NKT) lymphocytes. Aim: To determine whether b-glucosylceramide can alter NKT function in opposite directions, colitis was induced by intracolonic installation of trinitrobenzenesulphonic acid, and hepatocellular carcinoma (HCC) was induced by transplantation of Hep3B cells. Methods: The immunological effect of b-glucosylceramide was assessed by analysis of intrahepatic and intrasplenic lymphocyte populations, serum cytokines and STAT protein expression. Results: Administration of b-glucosylceramide led to alleviation of colitis and to suppression of HCC, manifested by improved survival and decreased tumour volume. The beneficial effects were associated with an opposite immunological effect in the two models: the peripheral:intrahepatic CD4:CD8 lymphocyte ratio increased in the colitis model and decreased in the HCC group. The peripheral:intrahepatic NKT lymphocyte ratio decreased in b-glucosylceramide-treated mice solely in the HCC model. The effect of b-glucosylceramide was associated with decreased STAT1 and 4 expression, and with overexpression of STAT6, along with decreased interferon c levels in the colitis model, whereas an opposite effect was noted in the HCC model. Conclusions: b-glucosylceramide alleviates immunologically incongruous disorders and may be associated with ''fine tuning'' of immune responses, by changes in plasticity of NKT lymphocytes.

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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-␥ 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.

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 x 10(6) 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.

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.

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 IFN␥ 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 mm 3 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 IFN␥ 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. Key words: hepatocellular carcinoma; dendritic cells; immunotherapy; natural killer T lymphocytesDendritic cells (DCs) are specialized antigen presenting cells (APCs) capable of taking up a diverse array of antigens and modulating T-cell immune response driving naive T-cells ...

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