Beta-glycosphingolipids improve glucose intolerance and hepatic steatosis of the Cohen diabetic rat

A link between altered levels of various gangliosides and the development of insulin resistance was described in transgenic mice. Naturally occurring glycosphingolipids were shown to exert immunomodulatory effects in a natural killer T (NKT) cell-dependent manner. This study examined whether glycosphingolipid-induced modulation of the immune system may reduce pancreatic and liver steatosis and stimulate insulin secretion in the Cohen diabetes-sensitive (CDS) rat, a lean model of non-insulin-resistant, nutritionally induced diabetes. Four groups of CDS rats fed a diabetogenic diet were treated with daily intraperitoneal injections of glycosphingolipids beta-glucosylceramide, beta-lactosylceramide, a combination of both (IGL), or vehicle (PBS) for up to 45 days. Immune modulation was assessed by fluorescence-activated cell sorting analysis of intrahepatic and intrasplenic lymphocytes. Steatosis was assessed by MRI imaging and histological examination of liver and pancreas, Blood glucose and plasma insulin concentrations were assessed during an oral glucose tolerance test. Administration of glycosphingolipids, particularly IGL, increased intrahepatic trapping of CD8 T and NKT lymphocytes. Pancreatic and liver histology were markedly improved and steatosis was reduced in all treated groups compared with vehicle-treated rats. Insulin secretion was restored after glycosphingolipid treatment, resulting in improved glucose tolerance. The immunomodulatory effect of beta-glycosphingolipids improved the beta-cell function of the hyperglycemic CDS rat. Thus our results suggest a role for the immune system in the pathogenesis of diabetes in this model.     

Selecting the optimal cell for kidney regeneration: fetal, adult or reprogrammed stem cells

Chronic kidney disease (CKD) is a progressive loss in renal function over a period of months or years. End-stage renal disease (ESRD) or stage 5 CKD ensues when renal function deteriorates to under 15% of the normal range. ESRD requires either dialysis or, preferentially, a kidney organ allograft, which is severely limited due to organ shortage for transplantation. To combat this situation, one needs to either increase supply of organs or decrease their demand. Two strategies therefore exist: for those that have completely lost their kidney function (ESRD), we will need to supply new kidneys. Taking into account the kidneys' extremely complex structure, this may prove to be impossible in the near future. In contrast, for those patients that are in the slow progression route from CKD to ESRD but still have functional kidneys, we might be able to halt progression by introducing stem cell therapy to diseased kidneys to rejuvenate or regenerate individual cell types. Multiple cell compartments that fall into three categories are likely to be worthy targets for cell repair: vessels, stroma (interstitium) and nephron epithelia. Different stem/progenitor cells can be linked to regeneration of specific cell types; hematopoietic progenitors and hemangioblastic cell types have specific effects on the vascular niche (vasculogenesis and angiogenesis). Multipotent stromal cells (MSC), whether derived from the bone marrow or isolated from the kidney's non-tubular compartment, may, in turn, heal nephron epithelia via paracrine mechanisms. Nevertheless, as we now know that all of the above lack nephrogenic potential, we should continue our quest to derive genuine nephron (epithelial) progenitors from differentiated pluripotent stem cells, from fetal and adult kidneys and from directly reprogrammed somatic cells.     

Induction of karyopherin α1 expression by indole-3-acetic acid in auxin-treated or overproducing tobacco plants

Macromolecules may transfer between the cytoplasm and the nucleus only through specific gates—the nuclear pore complexes (NPCs). Translocation of nucleic acids and large proteins requires the presence of a nuclear localization signal (NLS) within the transported molecule. This NLS is recognized by a class of soluble transport receptors termed karyopherins α and β. We previously characterized the expression pattern of the tomato karyopherin α1 (LeKAPα1) promoter in transformed tobacco plants. Expression of LeKAPα1 was mainly observed in growing tissues where cell division and extension is rapid. The expression pattern of LeKAPα1 resembled that of auxin-responsive genes. This led us to suggest that auxin participates in the regulation of LeKAPα1 expression. Here we characterized the correlation between auxin level and the activity of the LeKAPα1 promoter. To this end, transgenic tobacco plants carrying the GUS reporter gene under the control of the LeKAPα1 promoter were treated with various levels of exogenous auxin. We also studied transgenic plants in which we increased the endogenous levels of auxin. For this, we expressed in plants both the LeKAPα1 promoter-GUS reporter and the Agrobacterium tumefaciens iaaM gene, which increases the endogenous levels of auxin. The results indicate that the auxin indole-3-acetic acid (IAA) can induce LeKAPα1 expression. We also identified that the sites and levels of LeKAPα1 expression correlated with the endogenous pathways of polar auxin transport.Key words: auxin, karyopherin α1, nuclear pore complex, TYLCV, plant virus     

TGF-β1 Induced Transdifferentiation of RPE Cells is Mediated by TAK1

Background and Aim

Proliferative vitreoretinopathy (PVR) is an active process that develops as a complication upon retinal detachment (RD), accompanied by formation of fibrotic tissue. The main cells involved in the development of fibrotic tissue during PVR are the retinal pigment epithelial (RPE) cells. The RPE cells undergo epithelial-mesenchymal transition (EMT) which leads to complex retinal detachment and loss of vision. Transforming growth factor-β1 (TGF-β1) is considered as the main player in the EMT of RPE cells, even though the mechanism is not fully understood. This study was performed to determine the possible involvement of transforming growth factor β activated kinase 1 (TAK1) in the EMT process of the RPE cells.

Methodology

ARPE-19 Cells were treated with 5Z-7 oxozeaenol (TAK1 inhibitor) or SB431542 (TGF-β1 receptor kinase inhibitor) followed by TGF-β1 stimulation. Immunofluorescence, scratch assay Real time PCR and collagen contraction assay assessed the EMT features. The phosphorylation of Smad2/3 and p38 was examined using western blots analysis.

Results

This study demonstrates that stimulation of RPE cells with TGF-β1 increases α-SMA expression, cell migration and cell contractility, all of which are EMT features. Remarkably, addition of TAK1 inhibitor abolishes all these processes. Furthermore, we show hereby that TAK1 regulates not only the activation of the non-canonical cascade of TGF-β1 (p38), but also the canonical cascade, the Smad2/3 activation. Thus, the outcome of the TGF-β response in RPE cells is TAK1 dependent.

Conclusions/Significance

This work demonstrated TAK1, a component of the non-canonical pathway of TGF-β1, is a key player in the EMT process, thus provides deep insight into the pathogenesis of PVR. The ability to halt the process of EMT in RPE cells may reduce the severity of the fibrotic response that occurs upon PVR, leading to a better prognosis and increase the probability of success in RD treatment.     

Development of Allogeneic NK Cell Adoptive Transfer Therapy in Metastatic Melanoma Patients: In Vitro Preclinical Optimization Studies

Natural killer (NK) cells have long been considered as potential agents for adoptive cell therapy for solid cancer patients. Until today most studies utilized autologous NK cells and yielded disappointing results. Here we analyze various modular strategies to employ allogeneic NK cells for adoptive cell transfer, including donor-recipient HLA-C mismatching, selective activation and induction of melanoma-recognizing lysis receptors, and co-administration of antibodies to elicit antibody-dependent cell cytotoxicity (ADCC). We show that NK cell activation and induction of the relevant lysis receptors, as well as co-administration of antibodies yield substantial anti-cancer effects, which are functionally superior to HLA-C mismatching. Combination of the various strategies yielded improved effects. In addition, we developed various clinically-compatible ex vivo expansion protocols that were optimized according to fold expansion, purity and expression of lysis receptors. The main advantages of employing allogeneic NK cells are accessibility, the ability to use a single donor for many patients, combination with various strategies associated with the mechanism of action, e.g. antibodies and specific activation, as well as donor selection according to HLA or CD16 genotypes. This study rationalizes a clinical trial that combines adoptive transfer of highly potent allogeneic NK cells and antibody therapy.     

Association between Decreased Klotho Blood Levels and Organic Growth Hormone Deficiency in Children with Growth Impairment

Objective

Klotho is an aging-modulating protein expressed mainly in the kidneys and choroid plexus, which can also be shed, released into the circulation and act as a hormone. Klotho deficient mice are smaller compared to their wild-type counterparts and their somatotropes show marked atrophy and reduced number of secretory granules. Recent data also indicated an association between klotho levels and growth hormone (GH) levels in acromegaly. We aimed to study the association between klotho levels and GH deficiency (GHD) in children with growth impairment.

Design

Prospective study comprising 99 children and adolescents (aged 9.0±3.7 years, 49 male) undergoing GH stimulation tests for short stature (height-SDS = −2.1±0.6). Klotho serum levels were measured using an α-klotho ELISA kit.

Results

Klotho levels were significantly lower (p<0.001) among children with organic GHD (n = 11, 727±273 pg/ml) compared to both GH sufficient participants (n = 59, 1497±754 pg/ml) and those with idiopathic GHD (n = 29, 1645±778 pg/ml). The difference between GHS children and children with idiopathic GHD was not significant. Klotho levels positively correlated with IGF-1- standard deviation scores (SDS) (R = 0.45, p<0.001), but were not associated with gender, pubertal status, age or anthropometric measurements.

Conclusions

We have shown, for the first time, an association between low serum klotho levels and organic GHD. If validated by additional studies, serum klotho may serve as novel biomarker of organic GHD.     

Effect of carbon dioxide on neonatal mouse lung: a genomic approach.

Despite the deleterious effects associated with elevated carbon dioxide (CO(2)) or hypercapnia, it has been hypothesized that CO(2) can protect the lung from injury. However, the effects of chronic hypercapnia on the neonatal lung are unknown. Hence, we investigated the effect of chronic hypercapnia on neonatal mouse lung to identify genes that could potentially contribute to hypercapnia-mediated lung protection. Newborn mouse litters were exposed to 8% CO(2), 12% CO(2), or room air for 2 wk. Lungs were excised and analyzed for morphometric alterations. The alveolar walls of CO(2)-exposed mice appeared thinner than those of controls. Analyses of gene expression differences by microarrays revealed that genes from a variety of functional categories were differentially expressed following hypercapnia treatment, including those encoding growth factors, chemokines, cytokines, and endopeptidases. In particular and of major interest, the expression level of genes encoding surfactant proteins A and D, as well as chloride channel calcium-activated 3, were significantly increased, but the expression of WNT1-inducible signaling pathway protein 2 was significantly decreased. The significant changes in gene expression occurred mostly at 8% CO(2), but only a few at 12% CO(2). Our results lead us to conclude that 1) there are a number of gene families that may contribute to hypercapnia-mediated lung protection; 2) the upregulation of surfactant proteins A and D may play a role as anti-inflammatory or antioxidant agents; and 3) the effects of CO(2) seem to depend on the level to which the lung is exposed.     

Enhanced Levels of the Aroma and Flavor Compound S-Linalool by Metabolic Engineering of the Terpenoid Pathway in Tomato Fruits

The aromas of fruits, vegetables, and flowers are mixtures of volatile metabolites, often present in parts per billion levels or less. We show here that tomato (Lycopersicon esculentum Mill.) plants transgenic for a heterologous Clarkia breweri S-linalool synthase (LIS) gene, under the control of the tomato late-ripening-specific E8 promoter, synthesize and accumulate S-linalool and 8-hydroxylinalool in ripening fruits. Apart from the difference in volatiles, no other phenotypic alterations were noted, including the levels of other terpenoids such as gamma- and alpha-tocopherols, lycopene, beta-carotene, and lutein. Our studies indicate that it is possible to enhance the levels of monoterpenes in ripening fruits by metabolic engineering.