Systematic optimization of siRNA productive uptake into resting and activated T cellsex vivo
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Abstract RNA-based medicines are ideally suited for precise modulation of T cell phenotypes in anti-cancer immunity, in autoimmune diseases and for ex vivo modulation of T-cell-based therapies. Therefore, understanding productive siRNA uptake to T cells is of particular importance. Most studies used unmodified siRNAs or commercially available siRNA with undisclosed chemical modifications patterns to show functionality in T cells. Despite being an active field of research, robust siRNA delivery to T cells still represents a formidable challenge. Therefore, a systematic approach is needed to further optimize and understand productive siRNA uptake pathways to T cells. Here we compared conjugate-mediated and nanoparticle-mediated delivery of siRNAs to T cells in the context of fully chemically modified RNA constructs. We showed that lipid-conjugate-mediated delivery outperforms lipid-nanoparticle-mediated and extracellular-vesicle-mediated delivery in activated T cells ex vivo . Yet, ex vivo manipulation of T cells without the need of activation is of great therapeutic interest for CAR-T, engineered TCR-T and allogeneic donor lymphocyte applications. We are first to report productive siRNA uptake into resting T cells using lipid-conjugate mediated delivery. Interestingly, we observed strong dependence of silencing activity on lipid-conjugate-identity in resting T cells but not in activated T cells. This phenomenon is consistent with our early uptake kinetics data. Lipid-conjugates also enabled delivery of siRNA to all mononuclear immune cell types, including both lymphoid and myeloid lineages. These findings are expected to be broadly applicable for ex vivo modulation of immune cell therapies.Keywords:
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Мета. Покращення результатів лікування хворих з місцево-розповсюдженими та рецидивними солідними пухлинами черевної порожнини та заочеревинного простору.
Матеріали і методи. За період з червня 2015 по січень 2018 р. в Національному інституті раку виконали комбіновані оперативні втручання з нефректомією 28 хворим з приводу первинних місцево-розповсюджених та рецидивних солідних пухлин черевної порожнини та заочеревинного простору.
Результати. У 5 із 28 пацієнтів виконали нефректомію ex vivo ex situ з аутотрансплантацією нирки, у 4 - успішно. Гостре ушкодження нирок спостерігали у 6 (26%) хворих, яким аутотрансплантації нирки не виконували. Після операції померли 2 (8,7%) хворих. У пацієнтів, яким нирка була збережена, не спостерігали гострого ушкодження нирок, ніхто з цих пацієнтів не помер.
Висновки. З метою профілактики розвитку гострого ушкодження та хронічної хвороби нирок у майбутньому можливість виконання аутотрансплантації нирки у разі хірургічного лікування солідних пухлин черевної порожнини та заочеревинного простору, окрім первинного раку нирки, повинна бути розглянута щодо кожного хворого. Дану процедуру доцільно виконувати в спеціалізованих лікувальних закладах, де накопичено досвід в онковаскулярній хірургії.
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No AccessJournal of UrologyInvestigative Urology1 Feb 2010In Vitro, Ex Vivo and In Vivo Isotherms for Renal Cryotherapyis companion ofUnintended Consequences of Laparoscopic Surgery on Partial Nephrectomy for Kidney Cancer Jennifer L. Young, Surendra B. Kolla, Donald L. Pick, Petros Sountoulides, Oskar G. Kaufmann, Cervando G. Ortiz-Vanderdys, Victor B. Huynh, Adam G. Kaplan, Lorena A. Andrade, Kathryn E. Osann, Michael K. Louie, Elspeth M. McDougall, and Ralph V. Clayman Jennifer L. YoungJennifer L. Young , Surendra B. KollaSurendra B. Kolla , Donald L. PickDonald L. Pick , Petros SountoulidesPetros Sountoulides , Oskar G. KaufmannOskar G. Kaufmann , Cervando G. Ortiz-VanderdysCervando G. Ortiz-Vanderdys , Victor B. HuynhVictor B. Huynh , Adam G. KaplanAdam G. Kaplan , Lorena A. AndradeLorena A. Andrade , Kathryn E. OsannKathryn E. Osann , Michael K. LouieMichael K. Louie , Elspeth M. McDougallElspeth M. McDougall , and Ralph V. ClaymanRalph V. Clayman View All Author Informationhttps://doi.org/10.1016/j.juro.2009.09.072AboutFull TextPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract Purpose: Preoperative planning for renal cryotherapy is based on isotherms established in gel. We replicated gel isotherms and correlated them with ex vivo and in vivo isotherms in a porcine model. Materials and Methods: PERC-17 CryoProbes™ (1.7 mm) and IceRods™ (1.47 mm) underwent trials in gel, ex vivo and in vivo porcine kidneys. Temperatures were recorded at 13 predetermined locations with multipoint thermal sensors. Results: At the cryoprobe temperatures were not significantly different along the probe in any medium for either system (p = 0.0947 to 0.9609). However, away from the probe ex vivo and in vivo trials showed warmer temperatures toward the cryoprobe tip for each system (p = 0.0003 to 0.2141). Mean ± SE temperature 5 mm distal to the cryoprobe tip in vivo was 19.2C ± 16.1C for CryoProbes and 27.3C ± 11.2C for IceRods. Temperatures were consistently colder with CryoProbes than with IceRods in gel (p <0.00005), ex vivo (p <0.00005) and in vivo (p = 0.0014). At almost all sites temperatures were significantly colder in gel and in ex vivo kidney than in in vivo kidney for CryoProbes (p = 0.0107 and 0.0008, respectively) and for IceRods (each p <0.00005). Conclusions: Gel and ex vivo isotherms do not predict the in vivo pattern of freezing. Thus, they should not be used for preoperative planning. The cryoprobe should be passed 5 mm beyond the tumor border to achieve suitably cold temperatures. Multipoint thermal sensor probes are recommended to record actual temperature during renal cryotherapy. References 1 : Global increases in kidney cancer incidence, 1973–1992. Eur J Cancer Prev2002; 11: 171. Google Scholar 2 : The natural history of incidentally detected small renal masses. 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J Endourol2001; 15: 193. Google Scholar 24 : Temperature measurements of the low-attenuation radiographic ice ball during CT-guided renal cryoablation. Cardiovasc Intervent Radiol2008; 31: 116. Google Scholar University of California-Irvine, Orange, California© 2010 by American Urological AssociationFiguresReferencesRelatedDetailsRelated articlesJournal of Urology14 Dec 2009Unintended Consequences of Laparoscopic Surgery on Partial Nephrectomy for Kidney Cancer Volume 183Issue 2February 2010Page: 752-758 Advertisement Copyright & Permissions© 2010 by American Urological AssociationKeywordstemperaturekidney neoplasmsinstrumentationcryosurgerykidneyMetricsAuthor Information Jennifer L. Young More articles by this author Surendra B. Kolla More articles by this author Donald L. Pick More articles by this author Petros Sountoulides More articles by this author Oskar G. Kaufmann More articles by this author Cervando G. Ortiz-Vanderdys More articles by this author Victor B. Huynh More articles by this author Adam G. Kaplan More articles by this author Lorena A. Andrade More articles by this author Kathryn E. Osann More articles by this author Michael K. Louie More articles by this author Elspeth M. McDougall More articles by this author Ralph V. Clayman More articles by this author Expand All Advertisement PDF downloadLoading ...
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A few years ago, a promising optical device was created that could aid in the intraoperative localization of tumor tissue. Its discriminatory capacity for colorectal cancer tissue was investigated at the NKI-AVL. Measurements on healthy and malignant tissue were performed in and ex vivo. It was proposed to continue with only ex vivo measurements. Advantages of this approach were an increased reproducibility between measurements, easier inclusion and planning, and no need to interfere with the surgical procedure. The aim of this investigation was to validate whether the ex vivo measurements are comparable to in vivo measurements. A literature study showed that there is no clear consensus on the exact differences between in and ex vivo optical measurements (of colorectal tissues). From our statistical analysis and comparison of spectra, significant differences were found in the ex vivo measurements compared to in vivo measurements. These were mainly attributed to the loss of blood and water. The effects of these changes could be minimized if only the near-infrared (NIR) part of the spectrum is used. The fat parameters were uninfluenced in ex vivo measurements. It is therefore recommended to focus on fat and water dependent parameters obtained from NIR spectra.
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Abstract In vivo data acquisition using fiberoptic diffuse reflectance spectroscopy (DRS) is more complicated and less controlled compared to ex vivo data acquisition. It would be of great benefit if classifiers for in vivo tissue discrimination based on DRS could be trained on data obtained ex vivo. In this study, in vivo and ex vivo DRS measurements are obtained during colorectal cancer surgery. A mixed model statistical analysis is used to examine the differences between the two datasets. Furthermore, classifiers are trained and tested using in vivo and ex vivo data. It is found that with a classifier trained on ex vivo data and tested on in vivo data, similar results are obtained compared to a classifier trained and tested on in vivo data. In conclusion, under the conditions used in this study, classifiers intended for in vivo tissue discrimination can be trained on ex vivo data.
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Combinations of in vitro, ex vivo and in vivo animal testing have historically played a vital role in the research and the development of new therapeutic treatments. However, understanding the translation of these data across assays, as well as how these data translate to humans has proven to be challenging, especially with regards to the complex and integrated mechanism involved in blood pressure (BP) and heart rate (HR) regulation. We have evaluated the translation of several in vitro and ex vivo assays to in vivo CV assessments, as well as the translation of rat to large animal (LA), and LA to human BP and HR data. To investigate the translation of in vitro and ex vivo findings to in vivo hemodynamic results, 41 compounds with direct, indirect or unknown mechanisms of BP and/or HR modulation as well as 13 negative controls were evaluated in ppMLC (phosphorylation of myosin light chain), functional pharmacology (19 receptors/ion channels), ex vivo aortic vascular reactivity and isolated guinea pig Langendorff heart assays. Results from the in vitro and ex vivo assays were compared to in vivo conscious rat telemetry BP and HR findings. Vascular aortic relaxation and the paced guinea pig isolated heart assay detected 40% and 35%, respectively, of the in vivo positive compounds within 10× of the free Cmax that produced a 5 mmHg change in BP. The guinea pig isolated heart assay had a lower false positive rate than the aortic assay. The ppMLC assay detected 30% of compounds targeted at kinases within 10× the 5 mmHg in vivo Cmax concentration, but did not identify compounds with BP/HR risk with non‐kinase primary targets. Additional translation analyses were performed to more fully understand in vivo rat to large animal hemodynamic changes, as well as large animal preclinical to phase 1 clinical BP and HR measures. 83 compounds were assessed in both rat and LA studies. The specificity observed (true positive rate) was 71% and sensitivity (true negative rate) was 84%. Suggesting that rat is an effective model for evaluating hemodynamic risk. In evaluating 79 compounds in both LA and preclinical Phase 1 trials there was also good specificity (79%) and sensitivity (78%). These studies demonstrate that several in vitro/ex vivo assays are useful for identifying in vivo BP and HR changes; however due to the complexity of the CV system, have limited predictivity. Preclinical in vivo BP and HR testing was demonstrated to translate not only across species but also to the clinic. These data confirm the importance of pre‐clinical in vitro, ex vivo and animal models in the examination of the efficacy and safety of drugs, and the usefulness of these models for selection of compounds for advancement to clinical testing and ultimately the treatment of disease.
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