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organoid infection

Liang Tao, Jie Zhang, Paul Meraner, Alessio Tovaglieri, Xiaoqian Wu, Ralf Gerhard, Xinjun Zhang, William B Stallcup, Ji Miao, Xi He, Julian G Hurdle, David T Breault, Abraham L Brass, Min Dong
Clostridium difficile toxin B (TcdB) is a critical virulence factor that causes diseases associated with C. difficile infection. Here we carried out CRISPR-Cas9-mediated genome-wide screens and identified the members of the Wnt receptor frizzled family (FZDs) as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity towards FZD1, 2 and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signalling. FZD1/2/7 triple-knockout cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium...
September 28, 2016: Nature
Sina Bartfeld
Advances in stem cell research have allowed the development of 3-dimensional (3D) primary cell cultures termed organoid cultures, as they closely mimic the in vivo organization of different cell lineages. Bridging the gap between 2-dimensional (2D) monotypic cancer cell lines and whole organisms, organoids are now widely applied to model development and disease. Organoids hold immense promise for addressing novel questions in host-microbe interactions, infectious diseases and the resulting inflammatory conditions...
September 14, 2016: Developmental Biology
Walter Chingwaru, Richard H Glashoff, Jerneja Vidmar, Petrina Kapewangolo, Samantha L Sampson
Mycobacterium tuberculosis and human immunodeficiency virus (HIV) co-infections have remained a major public health concern worldwide, particularly in Southern Africa. Yet our understanding of the molecular interactions between the pathogens has remained poor due to lack of suitable preclinical models for such studies. We reviewed the use, this far, of mammalian cell culture models in HIV-MTB interaction studies. Studies have described the use of primary human cell cultures, including (1) monocyte-derived macrophage (MDM) fractions of peripheral blood mononuclear cell (PBMC), alveolar macrophages (AM), (2) cell lines such as the monocyte-derived macrophage cell line (U937), T lymphocyte cell lines (CEMx174, ESAT-6-specific CD4(+) T-cells) and an alveolar epithelial cell line (A549) and (3) special models such as stem cells, three dimensional (3D) or organoid cell models (including a blood-brain barrier cell model) in HIV-MTB interaction studies...
September 2016: Asian Pacific Journal of Tropical Medicine
Jessica L Forbester, Nicholas Hannan, Ludovic Vallier, Gordon Dougan
Intestinal human organoids (iHOs) provide an effective system for studying the intestinal epithelium and its interaction with various stimuli. By using combinations of different signaling factors, human induced pluripotent stem cells (hIPSCs) can be driven to differentiate down the intestinal lineage. Here, we describe the process for this differentiation, including the derivation of hindgut from hIPSCs, embedding hindgut into a pro-intestinal culture system and passaging the resulting iHOs. We then describe how to carry out microinjections to introduce bacteria to the apical side of the intestinal epithelial cells (IECs)...
August 31, 2016: Methods in Molecular Biology
Miao Xu, Emily M Lee, Zhexing Wen, Yichen Cheng, Wei-Kai Huang, Xuyu Qian, Julia Tcw, Jennifer Kouznetsova, Sarah C Ogden, Christy Hammack, Fadi Jacob, Ha Nam Nguyen, Misha Itkin, Catherine Hanna, Paul Shinn, Chase Allen, Samuel G Michael, Anton Simeonov, Wenwei Huang, Kimberly M Christian, Alison Goate, Kristen J Brennand, Ruili Huang, Menghang Xia, Guo-Li Ming, Wei Zheng, Hongjun Song, Hengli Tang
In response to the current global health emergency posed by the Zika virus (ZIKV) outbreak and its link to microcephaly and other neurological conditions, we performed a drug repurposing screen of ∼6,000 compounds that included approved drugs, clinical trial drug candidates and pharmacologically active compounds; we identified compounds that either inhibit ZIKV infection or suppress infection-induced caspase-3 activity in different neural cells. A pan-caspase inhibitor, emricasan, inhibited ZIKV-induced increases in caspase-3 activity and protected human cortical neural progenitors in both monolayer and three-dimensional organoid cultures...
October 2016: Nature Medicine
Julia Matheson, Claudia Bühnemann, Emma J Carter, David Barnes, Hans-Jürgen Hoppe, Jennifer Hughes, Stephen Cobbold, James Harper, Hans Morreau, Mirvat Surakhy, A Bassim Hassan
Two important protein-protein interactions establish E-cadherin (Cdh1) in the adhesion complex; homophilic binding via the extra-cellular (EC1) domain and cytoplasmic tail binding to β-catenin. Here, we evaluate whether E-cadherin binding can inhibit β-catenin when there is loss of Adenomatous polyposis coli (APC) from the β-catenin destruction complex. Combined conditional loss of Cdh1 and Apc were generated in the intestine, intestinal adenoma and adenoma organoids. Combined intestinal disruption (Cdh1fl/flApcfl/flVil-CreERT2) resulted in lethality, breakdown of the intestinal barrier, increased Wnt target gene expression and increased nuclear β-catenin localization, suggesting that E-cadherin inhibits β-catenin...
August 23, 2016: Oncotarget
Yong-Guo Zhang, Jun Sun
The intestinal epithelial cells function to gain nutrients, retain water and electrolytes, and form an efficient barrier against foreign microbes and antigens. Researchers employed cell culture lines derived from human or animal cancer cells as experimental models in vitro for understanding of intestinal infections. However, most in vitro models used to investigate interactions between bacteria and intestinal epithelial cells fail to recreate the differentiated tissue components and structure observed in the normal intestine...
August 19, 2016: Methods in Molecular Biology
Asako Tajima, Isha Pradhan, Massimo Trucco, Yong Fan
The thymus is the primary site for the generation of a diverse repertoire of T-cells that are essential to the efficient function of adaptive immunity. Numerous factors varying from aging, chemotherapy, radiation exposure, virus infection and inflammation contribute to thymus involution, a phenomenon manifested as loss of thymus cellularity, increased stromal fibrosis and diminished naïve T-cell output. Rejuvenating thymus function is a challenging task since it has limited regenerative capability and we still do not know how to successfully propagate thymic epithelial cells (TECs), the predominant population of the thymic stromal cells making up the thymic microenvironment...
June 2016: Current Stem Cell Reports
Isaac H Solomon, Danny A Milner, Rebecca D Folkerth
Zika virus (ZIKV) is a member of the Flaviviridae family that had been associated only with mild disease prior to the 2015 outbreak in Brazil. A dramatic increase in reported cases of microcephaly and Guillain-Barré syndrome during this time prompted significant research into possible associations with ZIKV and its neurotropic properties. Infection of neural progenitor cells and organoids have been shown to induce apoptosis and dysregulation of growth, and mouse studies have demonstrated viral replication in brain tissue in adults, as well as vertical transmission resulting in embryonic brain abnormalities...
June 2016: Journal of Neuroinfectious Diseases
Yuebang Yin, Yijin Wang, Wen Dang, Lei Xu, Junhong Su, Xinying Zhou, Wenshi Wang, Krzysztof Felczak, Luc J W van der Laan, Krzysztof W Pankiewicz, Annemiek A van der Eijk, Marcel Bijvelds, Dave Sprengers, Hugo de Jonge, Marion P G Koopmans, Herold J Metselaar, Maikel P Peppelenbosch, Qiuwei Pan
Rotavirus infection has emerged as an important cause of complications in organ transplantation recipients. Immunosuppressants used to prevent alloreactivity can also interfere with virus infection, but the direct effects of the specific type of immunosuppressants on rotavirus infection are still unclear. Here we profiled the effects of different immunosuppressants on rotavirus using a 2D culture model of Caco2 human intestinal cell line and a 3D model of human primary intestinal organoids inoculated with laboratory and patient-derived rotavirus strains...
September 2016: Antiviral Research
Jennifer Quantius, Carole Schmoldt, Ana I Vazquez-Armendariz, Christin Becker, Elie El Agha, Jochen Wilhelm, Rory E Morty, István Vadász, Konstantin Mayer, Stefan Gattenloehner, Ludger Fink, Mikhail Matrosovich, Xiaokun Li, Werner Seeger, Juergen Lohmeyer, Saverio Bellusci, Susanne Herold
Influenza Virus (IV) pneumonia is associated with severe damage of the lung epithelium and respiratory failure. Apart from efficient host defense, structural repair of the injured epithelium is crucial for survival of severe pneumonia. The molecular mechanisms underlying stem/progenitor cell mediated regenerative responses are not well characterized. In particular, the impact of IV infection on lung stem cells and their regenerative responses remains elusive. Our study demonstrates that a highly pathogenic IV infects various cell populations in the murine lung, but displays a strong tropism to an epithelial cell subset with high proliferative capacity, defined by the signature EpCamhighCD24lowintegrin(α6)high...
June 2016: PLoS Pathogens
Fernanda R Cugola, Isabella R Fernandes, Fabiele B Russo, Beatriz C Freitas, João L M Dias, Katia P Guimarães, Cecília Benazzato, Nathalia Almeida, Graciela C Pignatari, Sarah Romero, Carolina M Polonio, Isabela Cunha, Carla L Freitas, Wesley N Brandão, Cristiano Rossato, David G Andrade, Daniele de P Faria, Alexandre T Garcez, Carlos A Buchpigel, Carla T Braconi, Erica Mendes, Amadou A Sall, Paolo M de A Zanotto, Jean Pierre S Peron, Alysson R Muotri, Patricia C B Beltrão-Braga
Zika virus (ZIKV) is an arbovirus belonging to the genus Flavivirus (family Flaviviridae) and was first described in 1947 in Uganda following blood analyses of sentinel Rhesus monkeys. Until the twentieth century, the African and Asian lineages of the virus did not cause meaningful infections in humans. However, in 2007, vectored by Aedes aegypti mosquitoes, ZIKV caused the first noteworthy epidemic on the Yap Island in Micronesia. Patients experienced fever, skin rash, arthralgia and conjunctivitis. From 2013 to 2015, the Asian lineage of the virus caused further massive outbreaks in New Caledonia and French Polynesia...
May 11, 2016: Nature
Yoshiaki Maru, Kaoru Orihashi, Yoshitaka Hippo
Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids...
2016: Methods in Molecular Biology
Jason Dang, Shashi Kant Tiwari, Gianluigi Lichinchi, Yue Qin, Veena S Patil, Alexey M Eroshkin, Tariq M Rana
Emerging evidence from the current outbreak of Zika virus (ZIKV) indicates a strong causal link between Zika and microcephaly. To investigate how ZIKV infection leads to microcephaly, we used human embryonic stem cell-derived cerebral organoids to recapitulate early stage, first trimester fetal brain development. Here we show that a prototype strain of ZIKV, MR766, efficiently infects organoids and causes a decrease in overall organoid size that correlates with the kinetics of viral copy number. The innate immune receptor Toll-like-Receptor 3 (TLR3) was upregulated after ZIKV infection of human organoids and mouse neurospheres and TLR3 inhibition reduced the phenotypic effects of ZIKV infection...
August 4, 2016: Cell Stem Cell
Xuyu Qian, Ha Nam Nguyen, Mingxi M Song, Christopher Hadiono, Sarah C Ogden, Christy Hammack, Bing Yao, Gregory R Hamersky, Fadi Jacob, Chun Zhong, Ki-Jun Yoon, William Jeang, Li Lin, Yujing Li, Jai Thakor, Daniel A Berg, Ce Zhang, Eunchai Kang, Michael Chickering, David Nauen, Cheng-Ying Ho, Zhexing Wen, Kimberly M Christian, Pei-Yong Shi, Brady J Maher, Hao Wu, Peng Jin, Hengli Tang, Hongjun Song, Guo-Li Ming
Cerebral organoids, three-dimensional cultures that model organogenesis, provide a new platform to investigate human brain development. High cost, variability, and tissue heterogeneity limit their broad applications. Here, we developed a miniaturized spinning bioreactor (SpinΩ) to generate forebrain-specific organoids from human iPSCs. These organoids recapitulate key features of human cortical development, including progenitor zone organization, neurogenesis, gene expression, and, notably, a distinct human-specific outer radial glia cell layer...
May 19, 2016: Cell
Patricia P Garcez, Erick Correia Loiola, Rodrigo Madeiro da Costa, Luiza M Higa, Pablo Trindade, Rodrigo Delvecchio, Juliana Minardi Nascimento, Rodrigo Brindeiro, Amilcar Tanuri, Stevens K Rehen
Since the emergence of Zika virus (ZIKV), reports of microcephaly have increased considerably in Brazil; however, causality between the viral epidemic and malformations in fetal brains needs further confirmation. We examined the effects of ZIKV infection in human neural stem cells growing as neurospheres and brain organoids. Using immunocytochemistry and electron microscopy, we showed that ZIKV targets human brain cells, reducing their viability and growth as neurospheres and brain organoids. These results suggest that ZIKV abrogates neurogenesis during human brain development...
May 13, 2016: Science
Tomasz J Nowakowski, Alex A Pollen, Elizabeth Di Lullo, Carmen Sandoval-Espinosa, Marina Bershteyn, Arnold R Kriegstein
The recent outbreak of Zika virus (ZIKV) in Brazil has been linked to substantial increases in fetal abnormalities and microcephaly. However, information about the underlying molecular and cellular mechanisms connecting viral infection to these defects remains limited. In this study we have examined the expression of receptors implicated in cell entry of several enveloped viruses including ZIKV across diverse cell types in the developing brain. Using single-cell RNA-seq and immunohistochemistry, we found that the candidate viral entry receptor AXL is highly expressed by human radial glial cells, astrocytes, endothelial cells, and microglia in developing human cortex and by progenitor cells in developing retina...
May 5, 2016: Cell Stem Cell
A Aoki-Yoshida, S Saito, S Fukiya, R Aoki, Y Takayama, C Suzuki, K Sonoyama
Administration of Lactobacillus rhamnosus GG (LGG) has been reported to be therapeutically effective against acute secretory diarrhoea resulting from the structural and functional intestinal mucosal lesions induced by rotavirus infection; however, the underlying mechanisms remain to be completely elucidated. Because Toll-like receptor 3 (TLR3) plays a key role in the innate immune responses following the recognition of rotavirus, the present study examined whether LGG influences TLR3 gene expression in murine small intestine ex vivo and in vivo...
June 2016: Beneficial Microbes
Soichiro Sue, Wataru Shibata, Eri Kameta, Takeshi Sato, Yasuaki Ishii, Hiroaki Kaneko, Haruo Miwa, Tomohiko Sasaki, Toshihide Tamura, Masaaki Kondo, Shin Maeda
BACKGROUND: Helicobacter pylori induces chronic inflammation and intestinal metaplasia (IM) through genetic and epigenetic changes and activation of intracellular signaling pathways that contribute to gastric carcinogenesis. However, the precise mechanism of IM in gastric carcinogenesis has not been fully elucidated. We previously found that intestine-specific homeobox (ISX) mRNA expression increased in organoids cultured from Helicobacter-infected mouse mucosa. In this study, we elucidate the role of ISX in the development of IM and gastric carcinogenesis...
October 2016: Journal of Gastroenterology
Sina Bartfeld, Hans Clevers
Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor...
2015: Journal of Visualized Experiments: JoVE
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