tbuckley/cabot-search huggingface dataset:
270. Nanoparticle-Mediated Rhodopsin cDNA But Not the Intron-Containing DNA Delivery Causes Transgene Silencing in a Rhodopsin Knockout Model
Authors: Zongchao Han, Min Zheng, Rajendra Mitra
Journal: Molecular Therapy (2015)

Gene expression is moderated by a multitude of patterns. The cross-talk between the native promoters and all the non-coding endogenous elements, such as transcription factors, enhancers and introns, are tightly controlled in a precise and real-time manner. The use of cDNA transgene often causes transgene silencing as over-expression or under-expression is known to affect protein function. In contrast, large genomic DNA transgene showed positionindependent, copy number-dependent expression of the transgene. Precise control of rhodopsin expression is critical in maintaining viable photoreceptors. In the present study, two forms of rhodopsin DNAs, an intron-containing short form genomic DNA and an intron-less cDNA, were cloned into an S/MAR vector driven by the 500 bp mouse opsin (MOP) promoter. The vectors were further compacted into nanoparticles (NPs) and subretinally injected into a rhodopsin knockout mouse (RKO) model at postnatal day 3. Following injection, DNA methylation and histone status of the specific regions (bacteria plasmid backbone, promoter, rhodopsin gene and S/MAR) in the vectors were evaluated at various time points. Persistent expression and phenotypic improvement were found in RKO mice up to 8 months after NP-mediated intron-containing delivery but not the NPs housing the intron-less cDNA delivery (data submitted for publication under review). In this study, our results further indicated that epigenetic silencing occurred in vector-mediated gene transfer, which were contributed by the plasmid backbone and the cDNA of the transgene, but not the intron-containing transgene as evaluated by DNA methylation specific PCR followed by DNA sequencing and chromatin immunoprocipitation (ChIP) assay with highly specific antibodies for euchromatin and heterochromatin. No toxicity or inflammation was found in the treated eyes as detected by both PCR for pro-inflammatory cytokines and immunohistochemistry using macrophage marker, F4/80. These results suggest that cDNA of transgene and bacteria backbone interfere with the host defense mechanism of DNA methylation-mediated transgene silencing through heterochratin-associated modification. While the benefits of including genomic elements may vary from gene to gene, our data suggest reconsider the current paradigm using cDNA as the standard in gene delivery cassettes. This paradigm shift has wide-ranging application for ocular gene therapy for retinal diseases or for which a genetic treatment is of interest. Further experiments are currently underway to determine whether inclusion of additional endogenous elements will provide added advantages in terms of physiologically relevant gene expression.
Three Hundred and Fifty Cases of Pediatric Allogeneic Hematopoietic Stem Cell Transplantation: Single Center Study in Korea.
Authors: Young-Shil Park, Pil-Sang Jang, Sangrhim Choi, Nak-Gyun Chung, Bin Cho, Dae-Chul Jeong, Hack-Ki Kim
Journal: Blood (2004)
Hepatic Toxicity After Reduced-Intensity Conditioning Allogeneic Stem Cell Transplantation: Incidence, Characteristics and Risk Factors In a Cohort of 452 Patients.
Authors: Pere Barba, Rodrigo Martino, jose Luis Pinana, Jose Antonio Perez-Simon, Francesc fernandez-Aviles, David Valcarcel, Montserrat Rovira, Lucia Lopez-Anglada, Silvana Novelli, Enric Carreras, Jorge Sierra
Journal: Blood (2010)
Use of MRI in the diagnosis of fetal brain abnormalities in utero (MERIDIAN): a multicentre, prospective cohort study
Authors: Paul D Griffiths, Michael Bradburn, Michael J Campbell, Cindy L Cooper, Ruth Graham, Deborah Jarvis, Mark D Kilby, Gerald Mason, Cara Mooney, Stephen C Robson, Allan Wailoo
Journal: The Lancet (2016)

In-utero MRI (iuMRI) has shown promise as an adjunct to ultrasound but the comparative diagnostic performance has been poorly defined. We aimed to assess whether the diagnostic accuracy and confidence of the prenatal diagnosis of fetal brain abnormalities is improved with iuMRI and assess the clinical impact and patient acceptability of iuMRI.We did a multicentre, prospective, cohort study in the UK, at 16 fetal medicine centres, of pregnant women aged 16 years or older whose fetus had a brain abnormality detected by ultrasound at a gestational age of 18 weeks or more, had no contraindications to iuMRI, and consented to enter the study. Women carrying a fetus suspected of having a brain anomaly on ultrasound had iuMRI done within 14 days of ultrasound. The findings were reviewed by two independent panels and used to estimate diagnostic accuracy and confidence by comparison with outcome diagnoses. Changes in diagnosis, prognosis, and clinical management brought about by iuMRI and patient acceptability were assessed.Participants were recruited between July 29, 2011, and Aug 31, 2014. The cohort was subdivided by gestation into the 18 weeks to less than 24 weeks fetus cohort (n=369) and into the 24 weeks or older fetus cohort (n=201). Diagnostic accuracy was improved by 23% (95% CI 18-27) in the 18 weeks to less than 24 weeks group and 29% (23-36) in the 24 weeks and older group (p<0·0001 for both groups). The overall diagnostic accuracy was 68% for ultrasound and 93% for iuMRI (difference 25%, 95% CI 21-29). Dominant diagnoses were reported with high confidence on ultrasound in 465 (82%) of 570 cases compared with 544 (95%) of 570 cases on iuMRI. IuMRI provided additional diagnostic information in 387 (49%) of 783 cases, changed prognostic information in at least 157 (20%), and led to changes in clinical management in more than one in three cases. IuMRI also had high patient acceptability with at least 95% of women saying they would have an iuMRI study if a future pregnancy were complicated by a fetal brain abnormality.iuMRI improves diagnostic accuracy and confidence for fetal brain anomalies and leads to management changes in a high proportion of cases. This finding, along with the high patient acceptability, leads us to propose that any fetus with a suspected brain abnormality on ultrasound should have iuMRI to better inform counselling and management decisions.National Institute for Health Research Health Technology Assessment programme.
608. In Silico Reconstructed Ancestral Adeno-Associated Viruses Transduce Mouse Anterior Segment
Authors: Ru Xiao, Li Wang, Livia Carvalho, Sarah Wassmer, Luk H. Vandenberghe
Journal: Molecular Therapy (2016)

Adeno-associated virus (AAV) has been widely used as a gene delivery vehicle in ocular gene therapy, especially for the treatment of inherited retinal diseases. It has been proven in animal models and humans that many AAVs can efficiently transduce different cell types in the retina. However, there are very few reports showing that single strand AAVs can successfully transduce the anterior segment of the eye. Some self-complementary AAVs, such as self- complementary AAV2 (scAAV2), have been shown to have robust transduction in the anterior segment cells of mouse, rat, and monkey. Unfortunately, the maximum package genome size of the self-complementary AAV is approximately 2.2kbp, which is less than half the package capacity of single strand AAV vectors. In previous studies, we have shown that an ancestral AAV, Anc80, which was developed in our lab by in silico ancestral sequence reconstruction, can achieve fast onset and long-term stable expression in retinal cells. In the present study, we have explored the tropism and transduction efficiency of the ancestral AAV vector (Anc80) in the anterior segment of mice eyes and compared it with some other contemporary AAVs. Three different AAV vectors (single strand AAV2-ssAAV2, scAAV2 and Anc80) carrying eGFP reporter gene driven by CMV promoter. The viral vectors were injected into wild type C57Bl/6 mouse eyes either via anterior chamber injection at a dose of 1×109 particles, or via intravitreal injection at a dose of 2×109 particles. At day 28 post- injection, mice eyeballs were collected and sectioned to examine GFP expression in the anterior segment by using confocal microscopy. Our data shows all three AAV vectors can transduce cornea, trabecular meshwork, and ciliary body by anterior chamber injection, as determined by GFP expression level and expression area in the cornea, Anc80 > ssAAV2 > scAAV2, and in the trabecular meshwork and ciliary body, Anc80 > scAAV2 > ssAAV2. Via intravitreal injection, GFP expression was detected in cornea and ciliary body in the Anc80 group, while no GFP expression in cornea or ciliary body was found in the ssAAV2 group. In Conclusion, anterior chamber or intravitreal injections of Anc80 efficiently transduce all components of the mouse anterior segment. These results will inform future studies that aim to deliver therapeutics in such diseases as glaucoma, which affect the cornea, iris, or ciliary body.
Abstract 4965: Multiple ubiquitin-conjugating enzymes modulate the ubiquitination and downregulation of the EGFR by the Cbl RING finger ubiquitin ligase
Authors: Ke Ma, Philip Ryan, Rachel Klevit, Stanley Lipkowitz
Journal: Cancer Research (2015)

Abstract Receptor tyrosine kinases (RTKs) are known drivers of malignant transformation. Many RTKs (e.g., EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. E3s such as the Cbl proteins confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the target proteins in collaboration with ubiquitin-conjugating enzymes (E2s). We used enzymatic and yeast two-hybrid assays to characterize the E2s that can interact with Cbl proteins. Using an in vitro E3 assay, we found that only the Ube2d family of E2s mediates autoubiquitination of the Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that Ube2e1, Ube2e2, Ube2e3, Ube2l3, Ube2u, Ube2w and Ube2z can interact with Cbl even though they do not support autoubiquitination of Cbl in the in vitro E3 assay. Among these E2s, three Ube2e family members and Ube2w bind to the RF domain of Cbl as demonstrated by the loss of interaction when the RF domain is disrupted. This suggests that Ube2e and Ube2w are relevant to the ubiquitination and degradation of substrates by Cbl. Knockdown of Ube2w decreases downregulation of EGFR in Hela cells. In the in vitro E3 assay we found that Ube2w can increase autoubiquitination of Cbl mediated by ube2d2. Surprisingly, we found that knockdown of Ube2e increases downregulation and ubiquitination of EGFR in HeLa cells. Mechanistically we found that three Ube2e members inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Further, we showed that Ube2e does not affect ubiquitin charging of Ube2d2 by the ubiquitin-activating enzyme (E1) in vitro. This suggests that Ube2e does not compete with Ube2d2 for the E1 under these conditions. Thus, our data suggest that Ube2e acts as a positive modulator of EGFR signaling by competing for Cbl with Ube2d2 and thus prevents ubiquitination and downregulation of the EGFR by Cbl in combination with Ube2d2. Together these data demonstrate that there is an E2 network which modulates the ubiquitination and downregulation of the EGFR by Cbl. Citation Format: Ke Ma, Philip Ryan, Rachel Klevit, Stanley Lipkowitz. Multiple ubiquitin-conjugating enzymes modulate the ubiquitination and downregulation of the EGFR by the Cbl RING finger ubiquitin ligase. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4965. doi:10.1158/1538-7445.AM2015-4965
NK Receptor (NKR)-Mediated Signaling Pathways in Cord Blood (CB) CD56dim Versus Peripheral Blood (PB) CD56dim NK Cells: Implication for Immaturity in Cord Blood NK CD56dim Innate Immunity
Authors: Nancy Day, Evan Shereck, Janet Ayello, Catherine McGuinn, Prakash Satwani, Duane Lewis, Carmella van de Ven, Ronald J. Wapner, Megan S Lim, Mitchell S. Cairo
Journal: Blood (2008)
Transfer of the Multidrug Resistance Gene, MDR-1, to Hematopoietic Progenitors from a Patient with Transformed Large Cell Lymphoma, and Demonstration of In Vivo Chemotherapy Protection with Dose Escalating Chemotherapy.
Authors: Pamela S. Becker, Ying Ping Yu, Ann Marie Ceredona, William V. Walsh, Judith L. O’Donnell, Christopher Baum, Lynn Kallander, Philip A. Lowry, T. J. Fitzgerald, Peter Westervelt, F. Marc Stewart
Journal: Blood (2004)
High-dose carmustine, etoposide, cytarabine, cyclophosphamide (BEAC) is feasible in an outpatient community setting for patients (PTS) with relapsed and high-risk non-hodgkin's lymphoma (NHL)
Authors: Alan Kritz, Romeo Mandanas, Luceli Cuasay, Kimberly Quiett, Loretta A Williams, Roy Beveridge, Gary Spitzer, Robert Rifkin
Journal: Blood (2000)
Circulating Erythrocyte-Derived Microparticles Are Associated with Coagulation Activation in Sickle Cell Disease
Authors: Eduard J. Beers van, M. C. Schaap, R.J. Berckmans, R. Nieuwland, A. Sturk, J.C.M. Meijers, Bart J. Biemond
Journal: Blood (2008)
Long-term results of chemoradiotherapy followed by amrubicin and cisplatin for limited-disease small cell lung cancer
Authors: Ikuo Sekine, Minako Sumi, Miyako Satouchi, Kayoko Tsujino, Makoto Nishio, Takuhiro Kozuka, Seiji Niho, Keiji Nihei, Nobuyuki Yamamoto, Hideyuki Harada, Satoshi Ishikura, Tomohide Tamura
Journal: Annals of Oncology (2015)
Abstract 184: miR-379/411 cluster regulates IL-18 and contributes to drug resistance in malignant pleural mesothelioma
Authors: Akihiko Miyanaga, Masahiro Seike, Kazuo Yamamoto, Susumu Takeuchi, Rintaro Noro, Yuji Minegishi, Kaoru Kubota, Akihiko Gemma
Journal: Cancer Research (2015)

Abstract Malignant pleural mesothelioma (MPM) is a rapidly fatal malignancy that is increasing in incidence in Japan. In this study, we performed gene and microRNA (miRNA) expres¬sion profiling to identify novel therapeutic targets in MPM cells. Based on relative sensitivities to pemetrexed (PEM) and the histone deacetylase (HDAC) inhibitor, vorinostat (SAHA), 211H cells were determined to be the only sensitive MPM cell line out of the 6 tested. On the same series of cell lines, we performed whole genome transcriptomic profiling via DNA microarrays and pathway analysis of the derived data. Of particular note, IL-18 gene expression levels were signifi¬cantly higher in the cell lines that were either drug resistant or displayed intermediate sensitivity, compared to the sensitive 211H cell line. Pathway analysis revealed IL-18 as an impor¬tant gene associated with drug sensitivity of MPM cells. A relationship between IL-18 overexpression and drug resistance was also observed following targeted assessment of 10 cyto¬kine genes using quantitative RT-PCR. miRNA expression profiles were evaluated in the MPM cell line panel in order to discern the mechanism of IL-18 induction in the drug-resistant lines. We found that miR-379 and miR-411 belonged to the same cluster of miRNAs located on chromosome 14q32 that commonly target the IL-18 gene. Luciferase reporter assays revealed that miR-379 and miR-411 directly target the IL-18 gene. Introduction of miR-379 plus miR-411, as well as IL-18 silencing, significantly suppressed the invasive capacity of MESO1 cells in vitro. Furthermore, the use of either PEM or SAHA together with miR-379 plus miR-411 mimics mediated increased sensitivity to these drugs in MESO1 cells. These results suggest that the miR-379/411 cluster may provide new therapeutic opportunities for advanced MPM patients, depending on the nature of IL-18 gene expression. Citation Format: Akihiko Miyanaga, Masahiro Seike, Kazuo Yamamoto, Susumu Takeuchi, Rintaro Noro, Yuji Minegishi, Kaoru Kubota, Akihiko Gemma. miR-379/411 cluster regulates IL-18 and contributes to drug resistance in malignant pleural mesothelioma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 184. doi:10.1158/1538-7445.AM2015-184
239. Induction of Allograft Immune Tolerance with Bioengineered Thymus Organoids
Authors: Asako Tajima, Isha Pradhan, Ipsita Baneijee, William A. Rudert, Massimo Trucco, Yong Fan
Journal: Molecular Therapy (2016)

One of the major obstacles in solid organ transplantation is to establish immune tolerance of allografts. While immunosuppressive drugs can prevent graft rejection to a certain degree, their efficacies are limited, impermanent, and associated with severe side effects. As a primary immune organ essential to adaptive immunity, the thymus continuously generates a diverse population of naïve T-cells, which can effectively react to invading pathogens, but remains unresponsive to self-antigens. Many factors (e.g. infection, irradiation, drug treatments, or aging) can irreversibly compromise thymic function, resulting in immune deficiency, cancer, autoimmunity and other immunodysregulatory diseases. While numerous efforts have been made to modulate/rejuvenate thymic function, manipulating the thymus, either in vitro or in vivo, proves to be difficult. The major challenge is to reproduce its unique extracellular matrix microenvironment that is critical for the survival and function of thymic epithelial cells (TECs). TECs are the predominant population of thymic stromal cells that are essential for T-cell lineage determination and maturation. TECs cultured in the traditional 2-D culture rapidly lose their molecular properties and fail to grow, which prohibits them from undergoing genetic modification. We have recently developed a thymus decellularization technique, which allows us to reconstruct a functional thymus organoid de novo with isolated TECs. The 3-D thymic scaffolds can support the survival of TECs in vitro, and maintain their unique molecular properties. When transplanted into athymic nude mice, the bioengineered thymus organoids could effectively promote the homing of lymphocyte progenitors and support the development of a diverse, self-tolerant T-cell repertoire. The thymus-reconstructed nude mice could promptly reject skin allografts, and were able to mount antigen-specific humoral responses against ovalbumin upon immunization. Notably, tolerance to allogeneic skin grafts could be achieved by transplanting thymus organoids constructed with either TECs co-expressing both syngeneic and allogenic MHCs, or mixtures of donor and recipient TECs. Our results demonstrate the technical feasibility of inducing donor-specific allogeneic tolerance with bioengineered thymus organoids and highlight the clinical implications of this thymus reconstruction technique in solid organ transplantation and regenerative medicine.
324. Successful Repeated Delivery of Helper-Dependent Adenoviral Vector Toachieve Efficient Long-Term Human CFTR Expression in Mouse Airwaythrough Transient Immunosuppression
Authors: Huibi Cao, Jim Hu
Journal: Molecular Therapy (2016)

Cystic Fibrosis (CF) is a common life-threatening disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial chloride channel. As a chronic, lifelong disease, CF should be best treated with a continue level of CFTR expression. Pulmonary gene therapy may ultimately cure the CF lung disease. Thus, efficient long-term expression of the delivered CFTR gene in targeted cell types is essential to achieve this goal either by repeated application or with a long-duration expression system. Even though novel approaches to target airway progenitor/stem cells with gene editing may be more attractive, the efficiency for targeting specific progenitor cells will still be a challenge in airway delivery. Repeated administration of viral vectors may be required to increase the percentage of cells to be targeted and to boost long-term therapeutic effects. However, immune responses to viral proteins are the greatest barrier to repeated vector administration. Here, we demonstrated that repeated transduction of human CFTR gene in mouse airway can be successfully achieved with helper-dependent adenoviral (HD-Ad) vector through transient immunosuppression. We showed previously that cyclophosphamide significantly enhanced human CFTR expression in mice received HD-Ad vector readministration and sustained expression through inhibition of host immune reactions. In this study, we examined cyclophosphamide effects on expression of the human CFTR in multiple rounds of HD-Ad vector delivery to mouse airways. Four group mice were nasally delivered with an HD-Ad-K18-CFTR vector at 1.5×1010 vector particles per mouse at day 0, day 70 and day 120. Two groups were treated with cyclophosphamide 6 hours before and 4 day and 8 days after vector delivery each time (except the last round of vector delivery for mice sacrificed at day 123). The other two groups without treatment were used as controls. One treated group and one control group were sacrificed at day 123, and the others at day 153 for detecting transgene expression and immune reaction. We found that cyclophosphamide has significantly improved the expression of CFTR (4.1 times higher) compared to the control group as determined by real-time qPCR 3 days after last delivery. And the CFTR expression level was 3.1 times higher 30 days after last vector transduction. Cyclophosphamide also significantly reduced the levels of antiadenoviral and neutralizing antibodies in both BALF and serum as well as prevented leukocyte infiltration in lung tissues. This data suggested that efficient repeated transduction of human CFTR gene with HD-Ad vector can be achieved by transient immunopuression in mouse lung.