High-Shear Granulation of Hygroscopic Probiotic-Encapsulated Skim Milk Powder: Effects of Moisture-Activation and Resistant Maltodextrin.

A fine, hygroscopic, and poorly flowable probiotic powder encapsulating Lactobacillus rhamnosus GG (LGG) was granulated using a high-shear granulation process, wherein a small amount of water (4%, w/w) was used for moisture-activation with or without 10% (w/w) resistant maltodextrin (RM). The process consisted of four steps; premixing, agglomeration, moisture absorption, and drying steps. The moisture content, water activity, and viable cell count were monitored during the granulation. The size, morphology, and flowability of the granules were determined. The powder was successfully converted to about 10-times-larger granules (mass mean diameter = 162-204 µm) by this process, and the granules had a 'snowball' morphology. The LGG cells were well preserved under the high-shear granulation conditions, and the viable cell count of the granules greatly exceeded the minimum therapeutic level recommended for probiotic powders. The addition of RM decreased the moisture content of the granules; improved cell resistance to drying stress; narrowed the particle size distribution, with reductions seen in both very fine and very large particles; and produced more flowable granules. Moisture sorption analysis and differential scanning calorimetry demonstrated that these positive effects of RM on granulation were primarily attributed to its water distribution ability rather than its glass transition-related binding ability.

Ghrelin reverses ductular reaction and hepatic fibrosis in a rodent model of cholestasis.

The orexigenic peptide ghrelin (Ghr) stimulates hunger signals in the hypothalamus via growth hormone secretagogue receptor (GHS-R1a). Gastric Ghr is synthetized as a preprohormone which is proteolytically cleaved, and acylated by a membrane-bound acyl transferase (MBOAT). Circulating Ghr is reduced in cholestatic injuries, however Ghr's role in cholestasis is poorly understood. We investigated Ghr's effects on biliary hyperplasia and hepatic fibrosis in Mdr2-knockout (Mdr2KO) mice, a recognized model of cholestasis. Serum, stomach and liver were collected from Mdr2KO and FVBN control mice treated with Ghr, des-octanoyl-ghrelin (DG) or vehicle. Mdr2KO mice had lower expression of Ghr and MBOAT in the stomach, and lower levels of circulating Ghr compared to WT-controls. Treatment of Mdr2KO mice with Ghr improved plasma transaminases, reduced biliary and fibrosis markers. In the liver, GHS-R1a mRNA was expressed predominantly in cholangiocytes. Ghr but not DG, decreased cell proliferation via AMPK activation in cholangiocytes in vitro. AMPK inhibitors prevented Ghr-induced FOXO1 nuclear translocation and negative regulation of cell proliferation. Ghr treatment reduced ductular reaction and hepatic fibrosis in Mdr2KO mice, regulating cholangiocyte proliferation via GHS-R1a, a G-protein coupled receptor which causes increased intracellular Ca2+ and activation of AMPK and FOXO1, maintaining a low rate of cholangiocyte proliferation.

Transferability of a modified embryonic stem cell test using a new endpoint for developmental neurotoxicity.

We developed and analyzed a new surrogate endpoint of the mouse embryonic stem cell test (EST) for developmental neurotoxicity. To determine the sensitivity, specificity, and transferability of the new endpoint, a pre-validation team from three independent laboratories optimized and standardized the protocol for neuronal differentiation of mouse embryonic stem cells (mESCs) by measuring the neuronal differentiation rates of mESCs under different culture conditions, such as the presence or absence of basic fibroblast growth factor (bFGF) in the growth media and varying lengths of culture. In addition, a component ratio of neuronal cells was measured by using flow cytometry analysis of ß-III tubulin (Tuj1)-positive cells and real-time polymerase chain reaction analysis of microtubule-associated protein 2 (MAP2) mRNA. Our results showed that the best growth was achieved by culturing mESCs for 12 d in N2B27 medium without bFGF or ascorbic acid. Lead (II) acetate and aroclor 1254 were used to test the usefulness of the new endpoint. When we used the known ID(50) values for lead (II) acetate in the EST model, it was classified as non-embryotoxic; however, when we used the new ID(50) values that we determined in this study, it was classified as weakly embryotoxic. Aroclor 1254 and penicillin G were also classified as weakly embryotoxic and non-embryotoxic compounds, respectively, when cardiac and neuronal differentiation ID(50) values were used. Therefore, our new surrogate endpoint for developmental neurotoxicity is not only sensitive and specific but also transferable among laboratories.

Differential cytotoxic effects of mono-(2-ethylhexyl) phthalate on blastomere-derived embryonic stem cells and differentiating neurons.

Potential applications of embryonic stem (ES) cells are not limited to regenerative medicine but can also include in vitro screening of various toxicants. In this study, we established mouse ES cell lines from isolated blastomeres of two-cell stage embryos and examined their potential use as an in vitro system for the study of developmental toxicity. Two ES cell lines were established from 69 blastomere-derived blastocysts (2.9%). The blastomere-derived ES (bm-ES) cells were treated with mono-(2-ethylhexyl) phthalate (MEHP) in an undifferentiated state or after directed differentiation into early neural cell types. We observed significantly decreased cell viability when undifferentiated bm-ES cells were exposed to a high dose of MEHP (1000 microM). The cytotoxic effects of MEHP were accompanied by increased DNA fragmentation, nuclear condensation, and activation of Caspase-3, which are biochemical and morphological features of apoptosis. Compared to undifferentiated bm-ES cells, considerably lower doses of MEHP (50 and 100 microM) were sufficient to induce cell death in early neurons differentiated from bm-ES cells. At the lower doses, the number of neural cells positive for the active form of Caspase-3 was greater than that for undifferentiated bm-ES cells. Thus, our data indicate that differentiating neurons are more sensitive to MEHP than undifferentiated ES cells, and that undifferentiated ES cells may have more efficient defense systems against cytotoxic stresses. These findings might contribute to the development of a new predictive screening method for assessment of hazards for developmental toxicity.