Wheat Consumption Aggravates Colitis in Mice via Amylase Trypsin Inhibitor–mediated Dysbiosis

Background & Aims Wheat has become the world's major staple and its consumption correlates with prevalence of noncommunicable disorders such as inflammatory bowel diseases. Amylase trypsin inhibitors (ATIs), a component of wheat, activate the intestine's innate immune response via toll-like receptor 4 (TLR4). We investigated the effects of wheat and ATIs on severity of colitis and fecal microbiota in mice. Methods C57BL/6 wild-type and Tlr4–/– mice were fed wheat- or ATI-containing diets or a wheat-free (control) diet and then given dextran sodium sulfate to induce colitis; we also studied Il10–/– mice, which develop spontaneous colitis. Changes in fecal bacteria were assessed by taxa-speci…

In vivo fate mapping with SCL regulatory elements identifies progenitors for primitive and definitive hematopoiesis in mice.

10 páginas, 6 figuras.-- et al.

Enhanced protection of C57 BL/6 vs Balb/c mice to melanoma liver metastasis is mediated by NK cells.

ABSTRACT The B16F10 murine melanoma cell line displays a low expression of MHC class I molecules favoring immune evasion and metastases in immunocompetent C57 BL/6 wild-type mice. Here, we generated metastases to the liver, an organ that is skewed towards immune tolerance, by intrasplenic injection of B16F10 cells in syngeneic C57 BL/6 compared to allogeneic Balb/c mice. Surprisingly, Balb/c mice, which usually display a pronounced M2 macrophage and Th2 T cell polarization, were ∼3 times more susceptible to metastasis than C57 BL/6 mice, despite a much higher M1 and Th1 T cell immune response. The anti-metastatic advantage of C57 BL/6 mice could be attributed to a more potent NK-cell mediat…

Instruction of haematopoietic lineage choices, evolution of transcriptional landscapes and cancer stem cell hierarchies derived from an AML1-ETO mouse model.

The t(8;21) chromosomal translocation activates aberrant expression of the AML1-ETO (AE) fusion protein and is commonly associated with core binding factor acute myeloid leukaemia (CBF AML). Combining a conditional mouse model that closely resembles the slow evolution and the mosaic AE expression pattern of human t(8;21) CBF AML with global transcriptome sequencing, we find that disease progression was characterized by two principal pathogenic mechanisms. Initially, AE expression modified the lineage potential of haematopoietic stem cells (HSCs), resulting in the selective expansion of the myeloid compartment at the expense of normal erythro- and lymphopoiesis. This lineage skewing was foll…

Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion

mRNA reprogramming results in the generation of genetically stable induced pluripotent stem (iPS) cells while avoiding the risks of genomic integration. Previously published mRNA reprogramming protocols have proven to be inconsistent and time-consuming and mainly restricted to fibroblasts, thereby demonstrating the need for a simple but reproducible protocol applicable to various cell types. So far there have been no published reports using mRNA to reprogram any cell type derived from human blood. Nonmodified synthetic mRNAs are immunogenic and activate cellular defense mechanisms, which can lead to cell death and inhibit mRNA translation upon repetitive transfection. Hence, to overcome RNA…

Establishment and characterization of a nontumorigenic cell line derived from a human hepatocellular adenoma expressing hepatocyte-specific markers.

In the present study the establishment and characterization of a nontumorigenic liver epithelial cell line (HACL-1) derived from a human hepatocellular adenoma is described. The HACL-1 cells have a finite life span (i.e., they proliferate for a period of 2 months and then senesce), show cell-cell contact inhibition, do not grow in soft agar, are not tumorigenic when injected in nude mice, and possess a normal diploid karyotype. The cultured cells resemble hepatocytes, but exhibit some features of dedifferentiation. At the ultrastructural level the cells are endowed with round or oval nuclei, abundant cytoplasmic organelles, and varying amounts of glycogen. The rough endoplasmic reticulum is…

Measurement of Lymphocyte Proliferation: Critical Analysis of Radioactive and Photometric Methods

Different methods of lymphocyte proliferation are compared to identify a non-radioactive alternative to 3H-thymidine-test. The enzymatic assays evaluating the turnover of mitochondrial dehydrogenases (MTT-test) and lysosomal hexosaminidase (NAG-test) proved not sensitive enough to substitute for 3H-thymidine incorporation. The incorporation of the nucleotide analog 5-bromodeoxyuridine (BrdU) can be exploited using an ELISA-system (enzyme linked immunosorbent assay) employing a monoclonal anti-BrdU antibody to measure cell proliferation. An optimized test protocol of the BrdU-ELISA which fulfills the requirements for a sensitive and practicable non-radioactive alternative to 3H-thymidine-tes…

Contact-dependent inhibition of growth of normal diploid human fibroblasts by plasma membrane glycoproteins.

Homeostasis in vivo is maintained by a highly complex network of positive and negative signals. At the cellular level, this regulatory microenvironment can be divided, in a simplified fashion, into two major compartments: the humoral compartment, including compounds such as hormones, growth factors and nutrients, and the contact-environment compartment, including cell-cell and cell-matrix interactions. At least in cultures of diploid, non-transformed cells, cell-cell and cell-matrix interactions have been shown to be of major importance for the regulation of growth as well as of differentiation. Although until now the glycoprotein involved in the contact-dependent inhibition of growth has n…

The role of wild-type and mutated N-ras in the malignant transformation of liver cells

In order to determine the role of N-ras overexpression and mutation in malignant liver cell transformation, wild-type and mutated N-ras were transfected into the rat liver epithelial cell line OC/CDE 22, and N-ras expression, growth kinetics, growth in soft agar, and tumorigenicity in vivo as well as the involvement of the mitogen-activated protein kinase (MAPK) signal transduction pathway in the expression of the malignant phenotype were analyzed. Although OC/CDE 22 cells transfected with wild-type N-ras showed a high expression of N-ras at the mRNA and protein levels, the cells did not grow in soft agar and were not tumorigenic in vivo. In contrast, OC/CDE 22 cells transfected with mutate…

Nanomedicine: In Vivo Gene-Silencing in Fibrotic Liver by siRNA-Loaded Cationic Nanohydrogel Particles (Adv. Healthcare Mater. 18/2015)

Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system

Background Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors. Methods To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)).…

P0419 : In vivo cell specific gene silencing in the liver using novel siRNA-loaded nanohydrogel particles

Tetracycline-controlled transgenic targeting from the SCL locus directs conditional expression to erythrocytes, megakaryocytes, granulocytes, and c-kit-expressing lineage-negative hematopoietic cells

The stem cell leukemia gene SCL, also known as TAL-1, encodes a basic helix-loop-helix transcription factor expressed in erythroid, myeloid, megakaryocytic, and hematopoietic stem cells. To be able to make use of the unique tissue-restricted and spatio-temporal expression pattern of the SCL gene, we have generated a knock-in mouse line containing the tTA-2S tetracycline transactivator under the control of SCL regulatory elements. Analysis of this mouse using different tetracycline-dependent reporter strains demonstrated that switchable transgene expression was restricted to erythrocytes, megakaryocytes, granulocytes, and, importantly, to the c-kit-expressing and lineage-negative cell fracti…

Endothelial Wnt/β-catenin signaling reduces vascularization, barrier breakdown and tumor growth in a mouse glioma model

Isolation, biochemical characterization, long-term culture, and phenotype modulation of oval cells from carcinogen-fed rats.

Oval cells are liver epithelial cells that proliferate during hepatocarcinogenesis and chemically induced severe liver injury. It has been suggested that these cells represent hepatic stem cells which might play an important role in the histogenesis of cholangiocellular as well as hepatocellular carcinomas. In order to test this hypothesis highly purified oval cell preparations and propagable oval cell lines are needed. In the present study the isolation, biochemical characterization, and long-term culture of oval cells from rats fed a choline-deficient/DL-ethionine-supplemented diet for 6, 14, or 22 weeks are described. The freshly isolated oval cells were gamma-glutamyltranspeptidase-posi…

Growth control in mammalian cells by cell-cell contacts.

Growth of normal diploid mammalian cells in vitro is strongly regulated by the actual cell density. Cell-cell contacts via specific plasma membrane glycoproteins whose glycan moieties interact with specific receptors has been found to be a main growth regulatory principle. Malignant growth is suggested to result from impaired function of these receptors.

In Vivo Gene-Silencing in Fibrotic Liver by siRNA-Loaded Cationic Nanohydrogel Particles

Cationic nanohydrogel particles loaded with anti-Col1α1 siRNA suppress collagen synthesis and deposition in fibrotic mice: Systemically administered 40 nm sized nanogel particles accumulate in collagen-expressing cells in the liver. Their siRNA payload induces a sequence specific in vivo gene knockdown affording an efficient antifibrotic effect in mice with liver fibrosis.

RNAi knock-down mice: an emerging technology for post-genomic functional genetics

RNA interference (RNAi) has been extensively used for sequence-specific silencing of gene function in mammalian cells. The latest major breakthrough in the application of RNAi technology came from experiments demonstrating RNAi-mediated gene repression in mice and rats. After more than two decades of functional mouse research aimed at developing and continuously improving transgenic and knock-out technology, the advent of RNAi knock-down mice represents a valuable new alternative for studying gene function in vivo. In this review we provide some basic insight as to how RNAi can induce gene silencing to then focus on recent findings concerning the applicability of RNAi for regulating gene fu…

Endothelial Wnt/β-catenin signaling inhibits glioma angiogenesis and normalizes tumor blood vessels by inducing PDGF-B expression

Wnt modulates glioma vascularization by regulating PDGF-B expression.