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Gerhardinger, Chiara

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Gerhardinger

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Chiara

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Gerhardinger, Chiara
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    Multiple knockout mouse models reveal lincRNAs are required for life and brain development
    (eLife Sciences Publications, Ltd, 2013) Sauvageau, Martin; Goff, Loyal; Lodato, Simona; Bonev, Boyan; Groff, Abigail F.; Gerhardinger, Chiara; Sanchez-Gomez, Diana B; Hacisuleyman, Ezgi; Li, Eric; Spence, Matthew; Liapis, Stephen C; Mallard, William; Morse, Michael; Swerdel, Mavis R; D’Ecclessis, Michael F; Moore, Jennifer C; Lai, Venus; Gong, Guochun; Yancopoulos, George D; Frendewey, David; Kellis, Manolis; Hart, Ronald P; Valenzuela, David M; Arlotta, Paola; Rinn, John
    Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc–Brn1b and linc–Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr−/− neonates, whereas linc–Brn1b−/− mutants displayed distinct abnormalities in the generation of upper layer II–IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001
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    In vivo Ebola virus infection leads to a strong innate response in circulating immune cells
    (BioMed Central, 2016) Caballero, Ignacio S.; Honko, Anna N.; Gire, Stephen K.; Winnicki, Sarah; Melé, Marta; Gerhardinger, Chiara; Lin, Aaron; Rinn, John; Sabeti, Pardis; Hensley, Lisa E.; Connor, John H.
    Background: Ebola virus is the causative agent of a severe syndrome in humans with a fatality rate that can approach 90 %. During infection, the host immune response is thought to become dysregulated, but the mechanisms through which this happens are not entirely understood. In this study, we analyze RNA sequencing data to determine the host response to Ebola virus infection in circulating immune cells. Results: Approximately half of the 100 genes with the strongest early increases in expression were interferon-stimulated genes, such as ISG15, OAS1, IFIT2, HERC5, MX1 and DHX58. Other highly upregulated genes included cytokines CXCL11, CCL7, IL2RA, IL2R1, IL15RA, and CSF2RB, which have not been previously reported to change during Ebola virus infection. Comparing this response in two different models of exposure (intramuscular and aerosol) revealed a similar signature of infection. The strong innate response in the aerosol model was seen not only in circulating cells, but also in primary and secondary target tissues. Conversely, the innate immune response of vaccinated macaques was almost non-existent. This suggests that the innate response is a major aspect of the cellular response to Ebola virus infection in multiple tissues. Conclusions: Ebola virus causes a severe infection in humans that is associated with high mortality. The host immune response to virus infection is thought to be an important aspect leading to severe pathology, but the components of this overactive response are not well characterized. Here, we analyzed how circulating immune cells respond to the virus and found that there is a strong innate response dependent on active virus replication. This finding is in stark contrast to in vitro evidence showing a suppression of innate immune signaling, and it suggests that the strong innate response we observe in infected animals may be an important contributor to pathogenesis. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3060-0) contains supplementary material, which is available to authorized users.
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    IL-1\(\beta\) Is Upregulated in the Diabetic Retina and Retinal Vessels: Cell-Specific Effect of High Glucose and IL-1\(\beta\) Autostimulation
    (Public Library of Science, 2012) Liu, Yang; Biarnés Costa, Montserrat; Gerhardinger, Chiara
    Many molecular and cellular abnormalities detected in the diabetic retina support a role for IL-1\(\beta\)-driven neuroinflammation in the pathogenesis of diabetic retinopathy. IL-1\(\beta\) is well known for its role in the induction and, through autostimulation, amplification of neuroinflammation. Upregulation of IL-1\(\beta\) has been consistently detected in the diabetic retina; however, the mechanisms and cellular source of IL-1\(\beta\) overexpression are poorly understood. The aim of this study was to investigate the effect of high glucose and IL-1\(\beta\) itself on IL-1\(\beta\) expression in microglial, macroglial (astrocytes and Müller cells) and retinal vascular endothelial cells; and to study the effect of diabetes on the expression of IL-1\(\beta\) in isolated retinal vessels and on the temporal pattern of IL-1\(\beta\) upregulation and glial reactivity in the retina of streptozotocin-diabetic rats. IL-1\(\beta\) was quantified by RealTime RT-PCR and ELISA, glial fibrillar acidic protein, \(\alpha\)2-macroglobulin, and ceruloplasmin by immunoblotting. We found that high glucose induced a 3-fold increase of IL-1\(\beta\) expression in retinal endothelial cells but not in macroglia and microglia. IL-1\(\beta\) induced its own synthesis in endothelial and macroglial cells but not in microglia. In retinal endothelial cells, the high glucose-induced IL-1\(\beta\) overexpression was prevented by calphostin C, a protein kinase C inhibitor. The retinal vessels of diabetic rats showed increased IL-1\(\beta\) expression as compared to non-diabetic rats. Retinal expression of IL-1\(\beta\) increased early after the induction of diabetes, continued to increase with progression of the disease, and was temporally associated with upregulation of markers of glial activation. These findings point to hyperglycemia as the trigger and to the endothelium as the origin of the initial retinal upregulation of IL-1\(\beta\) in diabetes; and to IL-1\(\beta\) itself, via autostimulation in endothelial and macroglial cells, as the mechanism of sustained IL-1\(\beta\) overexpression. Interrupting the vicious circle triggered by IL-1\(\beta\) autostimulation could limit the progression of diabetic retinopathy.
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    Decreased Cerebrovascular Brain-Derived Neurotrophic Factor–Mediated Neuroprotection in the Diabetic Brain
    (American Diabetes Association, 2011) Navaratna, Deepti; Guo, Shu-Zhen; Hayakawa, Kazhuhide; Wang, Xiaoying; Gerhardinger, Chiara; Lo, Eng
    Objective: Diabetes is an independent risk factor for stroke. However, the underlying mechanism of how diabetes confers that this risk is not fully understood. We hypothesize that secretion of neurotrophic factors by the cerebral endothelium, such as brain-derived neurotrophic factor (BDNF), is suppressed in diabetes. Consequently, such accrued neuroprotective deficits make neurons more vulnerable to injury. Research Design and Methods: We examined BDNF protein levels in a streptozotocin-induced rat model of diabetes by Western blotting and immunohistochemistry. Levels of total and secreted BDNF protein were quantified in human brain microvascular endothelial cells after exposure to advanced glycation end product (AGE)-BSA by enzyme-linked immunosorbent assay and immunocytochemistry. In media transfer experiments, the neuroprotective efficacy of conditioned media from normal healthy endothelial cells was compared with AGE-treated endothelial cells in an in vitro hypoxic injury model. Results: Cerebrovascular BDNF protein was reduced in the cortical endothelium in 6-month diabetic rats. Immunohistochemical analysis of 6-week diabetic brain sections showed that the reduction of BDNF occurs early after induction of diabetes. Treatment of brain microvascular endothelial cells with AGE caused a similar reduction in BDNF protein and secretion in an extracellular signal–related kinase-dependent manner. In media transfer experiments, conditioned media from AGE-treated endothelial cells were less neuroprotective against hypoxic injury because of a decrease in secreted BDNF. Conclusions: Taken together, our findings suggest that a progressive depletion of microvascular neuroprotection in diabetes elevates the risk of neuronal injury for a variety of central nervous system diseases, including stroke and neurodegeneration.
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    The Transforming Growth Factor-β Pathway is a Common Target of Drugs that Prevent Experimental Diabetic Retinopathy
    (American Diabetes Association, 2009) Gerhardinger, Chiara; Dagher, Zeina; Sebastiani, Paola; Park, Yong Seek; Lorenzi, Mara
    OBJECTIVE-- Prevention of diabetic retinopathy would benefit from availability of drugs that preempt the effects of hyperglycemia on retinal vessels. We aimed to identify candidate drug targets by investigating the molecular effects of drugs that prevent retinal capillary demise in the diabetic rat. RESEARCH DESIGN AND METHODS-- We examined the gene expression profile of retinal vessels isolated from rats with 6 months of streptozotocin-induced diabetes and compared it with that of control rats. We then tested whether the aldose reductase inhibitor sorbinil and aspirin, which have different mechanisms of action, prevented common molecular abnormalities induced by diabetes. The Affymetrix GeneChip Rat Genome 230 2.0 array was complemented by real-time RT-PCR, immunoblotting, and immunohistochemistry. RESULTS-- The retinal vessels of diabetic rats showed differential expression of 20 genes of the transforming growth factor (TGF)-β pathway, in addition to genes involved in oxidative stress, inflammation, vascular remodeling, and apoptosis. The complete loop of TGF-β signaling, including Smad2 phosphorylation, was enhanced in the retinal vessels, but not in the neural retina. Sorbinil normalized the expression of 71% of the genes related to oxidative stress and 62% of those related to inflammation. Aspirin had minimal or no effect on these two categories. The two drugs were instead concordant in reducing the upregulation of genes of the TGF-β pathway (55% for sorbinil and 40% for aspirin) and apoptosis (74 and 42%, respectively). CONCLUSIONS-- Oxidative and inflammatory stress is the distinct signature that the polyol pathway leaves on retinal vessels. TGF-β and apoptosis are, however, the ultimate targets to prevent the capillary demise in diabetic retinopathy.
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    Dynamic changes during the treatment of pancreatic cancer
    (Impact Journals LLC, 2018) Wolff, Robert A.; Wang-Gillam, Andrea; Alvarez, Hector; Tiriac, Hervé; Engle, Dannielle; Hou, Shurong; Groff, Abigail F.; San Lucas, Anthony; Bernard, Vincent; Allenson, Kelvin; Castillo, Jonathan; Kim, Dong; Mulu, Feven; Huang, Jonathan; Stephens, Bret; Wistuba, Ignacio I.; Katz, Matthew; Varadhachary, Gauri; Park, YoungKyu; Hicks, James; Chinnaiyan, Arul; Scampavia, Louis; Spicer, Timothy; Gerhardinger, Chiara; Maitra, Anirban; Tuveson, David; Rinn, John; Lizee, Gregory; Yee, Cassian; Levine, Arnold J.
    This manuscript follows a single patient with pancreatic adenocarcinoma for a five year period, detailing the clinical record, pathology, the dynamic evolution of molecular and cellular alterations as well as the responses to treatments with chemotherapies, targeted therapies and immunotherapies. DNA and RNA samples from biopsies and blood identified a dynamic set of changes in allelic imbalances and copy number variations in response to therapies. Organoid cultures established from biopsies over time were employed for extensive drug testing to determine if this approach was feasible for treatments. When an unusual drug response was detected, an extensive RNA sequencing analysis was employed to establish novel mechanisms of action of this drug. Organoid cell cultures were employed to identify possible antigens associated with the tumor and the patient’s T-cells were expanded against one of these antigens. Similar and identical T-cell receptor sequences were observed in the initial biopsy and the expanded T-cell population. Immunotherapy treatment failed to shrink the tumor, which had undergone an epithelial to mesenchymal transition prior to therapy. A warm autopsy of the metastatic lung tumor permitted an extensive analysis of tumor heterogeneity over five years of treatment and surgery. This detailed analysis of the clinical descriptions, imaging, pathology, molecular and cellular evolution of the tumors, treatments, and responses to chemotherapy, targeted therapies, and immunotherapies, as well as attempts at the development of personalized medical treatments for a single patient should provide a valuable guide to future directions in cancer treatment.
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    High‐throughput identification of RNA nuclear enrichment sequences
    (John Wiley and Sons Inc., 2018) Shukla, Chinmay; McCorkindale, Alexandra; Gerhardinger, Chiara; Korthauer, Keegan; Cabili, Moran N; Shechner, David M; Irizarry, Rafael; Maass, Philipp; Rinn, John
    Abstract In the post‐genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ever‐growing catalogs require high‐throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse. Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA‐based functionality. We applied MPRNA to identify RNA‐based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine‐rich motif. These nuclear enrichment sequences are highly conserved and over‐represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single‐molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA‐based functionalities.