Detailed spatial information of low-molecular-weight compounds distribution, especially in the brain, is crucial towards understanding their mechanism of actions. Imaging techniques that can directly visualize drugs in the brain at a high resolution will complement existing tools for drug distribution analysis. Here, we performed surface-enhanced Raman scattering (SERS) imaging using a bioorthogonal alkyne tag to visualize drugs directly in situ at a high resolution. Focusing on the selective serotonin reuptake inhibitor S-citalopram (S-Cit), which possesses a nitrile group, we substituted an alkynyl group into its structure and synthesized alkynylated S-Cit (Alk-S-Cit). The brain transitivity and the serotonin reuptake inhibition of Alk-S-Cit were not significantly different as compared to S-Cit. Alk-S-Cit was visualized in the coronal mouse brain section using SERS imaging with silver nanoparticles. Further, SERS imaging combined with fluorescence microscopy allowed Alk-S-Cit to be visualized in the adjacent neuronal membranes, and in the brain vessel and parenchyma. Thus, our multimodal imaging technique is an effective method for detecting low-molecular-weight compounds in their original tissue environment and can potentially offer additional information regarding the precise spatial distribution of such drugs.
Masato Tanuma, Atsushi Kasai, Kazuki Bando, Naoyuki Kotoku, Kazuo Harada, Masafumi Minoshima, Kosuke Higashino, Atsushi Kimishima, Masayoshi Arai, Yukio Ago, Kaoru Seiriki, Kazuya Kikuchi, Satoshi Kawata, Katsumasa Fujita, Hitoshi Hashimoto
Cell therapy raises high hopes for better treatment of brain disorders. However, the majority of transplanted cells often die soon after transplantation and those that survive initially continue to die in the subacute phase, diminishing the impact of transplantations. In this study, we genetically modified transplanted human neural stem cells (hNSCs), from two distant embryonic SCs lines (H9 and RC17) to express one of four prosurvival factors – Hif1a, Akt1, Bcl-2, or Bcl-xl – and studied how these modifications improve short- and long-term survival of transplanted hNSCs. All genetic modifications dramatically increased survival of the transplanted hNSCs. Importantly, three out of four modifications also enhanced the exit of hNSCs from the cell cycle, thus avoiding aberrant growth of the transplants. Bcl-xl expression provided the strongest protection of transplanted cells, reducing both immediate and delayed cell death, and stimulated hNSC differentiation towards neuronal and oligodendroglial lineages. By designing hNSCs with drug-controlled expression of Bcl-xl, we demonstrated that short-term expression of a prosurvival factor can ensure the long-term survival of transplanted cells. Importantly, transplantation of Bcl-xl expressing hNSCs into mice suffering from stroke improved behavioral outcome and recovery of motor activity in mice.
Irina Korshunova, Sina Rhein, Diego García-González, Ines Stölting, Ulrich Pfisterer, Anna Barta, Oksana Dmytriyeva, Agnete Kirkeby, Markus Schwaninger, Konstantin Khodosevich
Background. We hypothesized that dynamic perfluorinated gas magnetic resonance imaging (19F MRI) would sensitively detect mild cystic fibrosis (CF) lung. Methods. This prospective study enrolled 20 healthy volunteers and 24 stable subjects with CF, including a subgroup of subjects with normal FEV1 (>80% predicted, n = 9). Dynamic 19F MRI images were acquired during sequential breath holds while breathing perfluoropropane (PFP) and during gas wash-out. Outcomes included the fraction of lung without significant ventilation (ventilation defect percent, VDP) and time constants that described PFP wash-in and wash-out kinetics. Results. VDP values (mean ± SD) of healthy controls (3.87% ± 2.7%) were statistically different from moderate CF subjects (19.5% ± 15.5%, P = 0.001) but not from mild CF subjects (10.4% ± 9.9%, P = 0.24) . The fractional lung volume with slow gas wash-out was elevated both in subjects with mild (9.61% ± 4.87%; P = 0.0066) and moderate CF (16.01% ± 5.01%; P = 0.0002) when compared to healthy controls (3.84% ± 2.16%). Conclusion. 19F MRI detected significant ventilation abnormalities in subjects with cystic fibrosis. Assessment of gas wash-out kinetics was more sensitive to mild CF lung disease than quantitation of steady state ventilation defects making 19F MRI a potentially valuable method for the characterization of early lung disease in CF.
Jennifer L. Goralski, Sang Hun Chung, Tyler M. Glass, Agathe S. Ceppe, Esther O. Akinnagbe-Zusterzeel, Aaron T. Trimble, Richard C. Boucher, Brian J. Soher, H. Cecil Charles, Scott H. Donaldson, Yueh Z. Lee
While gene transfer using recombinant adeno-associated viral (rAAV) vectors has shown success in some clinical trials, there remain many tissues that are not well transduced. Because of the recent success in reprogramming islet-derived cells into functional β cells in animal models, we constructed 2 highly complex barcoded replication competent capsid shuffled libraries and selected for high-transducing variants on primary human islets. We describe the generation of a chimeric AAV capsid (AAV-KP1) that facilitates transduction of primary human islet cells and human embryonic stem cell–derived β cells with up to 10-fold higher efficiency compared with previously studied best-in-class AAV vectors. Remarkably, this chimeric capsid also enabled transduction of both mouse and human hepatocytes at very high levels in a humanized chimeric mouse model, thus providing a versatile vector that has the potential to be used in both preclinical testing and human clinical trials for liver-based diseases and diabetes.
Katja Pekrun, Gustavo De Alencastro, Qing-Jun Luo, Jun Liu, Youngjin Kim, Sean Nygaard, Feorillo Galivo, Feijie Zhang, Ren Song, Matthew R. Tiffany, Jianpeng Xu, Matthias Hebrok, Markus Grompe, Mark A. Kay
Islet transplantation is an effective therapy for achieving and maintaining normoglycemia in patients with type 1 diabetes mellitus. However, the supply of transplantable human islets is limited. Upon removal from the pancreas, islets rapidly disintegrate and lose function, resulting in a short interval for studies of islet biology and pretransplantation assessment. Here, we developed a biomimetic platform that can sustain human islet physiology for a prolonged period ex vivo. Our approach involved the creation of a multichannel perifusion system to monitor dynamic insulin secretion and intracellular calcium flux simultaneously, enabling the systematic evaluation of glucose-stimulated insulin secretion under multiple conditions. Using this tool, we developed a nanofibrillar cellulose hydrogel–based islet-preserving platform (iPreP) that can preserve islet viability, morphology, and function for nearly 12 weeks ex vivo, and with the ability to ameliorate glucose levels upon transplantation into diabetic hosts. Our platform has potential applications in the prolonged maintenance of human islets, providing an expanded time window for pretransplantation assessment and islet studies.
Yi-Ju Chen, Taiji Yamazoe, Karla F. Leavens, Fabian L. Cardenas-Diaz, Andrei Georgescu, Dongeun Huh, Paul Gadue, Ben Z. Stanger
Broadly neutralizing antibodies (bNAbs) against HIV-1 are under evaluation for both prevention and therapy. HIV-1 sequence diversity observed in most HIV-infected individuals and archived variations in critical bNAb epitopes present a major challenge for the clinical application of bNAbs, as preexistent resistant viral strains can emerge, resulting in bNAb failure to control HIV. In order to identify viral resistance in patients prior to antibody therapy and to guide the selection of effective bNAb combination regimens, we developed what we believe to be a novel Bayesian machine-learning model that uses HIV-1 envelope protein sequences and foremost approximated glycan occupancy information as variables to quantitatively predict the half-maximal inhibitory concentrations (IC50) of 126 neutralizing antibodies against a variety of cross clade viruses. We then applied this model to peripheral blood mononuclear cell–derived proviral Env sequences from 25 HIV-1–infected individuals mapping the landscape of neutralization resistance within each individual’s reservoir and determined the predicted ideal bNAb combination to achieve 100% neutralization at IC50 values <1 μg/ml. Furthermore, predicted cellular viral reservoir neutralization signatures of individuals before an analytical antiretroviral treatment interruption were consistent with the measured neutralization susceptibilities of the respective plasma rebound viruses, validating our model as a potentially novel tool to facilitate the advancement of bNAbs into the clinic.
Wen-Han Yu, David Su, Julia Torabi, Christine M. Fennessey, Andrea Shiakolas, Rebecca Lynch, Tae-Wook Chun, Nicole Doria-Rose, Galit Alter, Michael S. Seaman, Brandon F. Keele, Douglas A. Lauffenburger, Boris Julg
Solid tumors impose immunological and physical barriers to the efficacy of chimeric antigen receptor (CAR) T-cell therapy that are not reflected in conventional pre-clinical testing against singularized tumor cells in two-dimensional culture. Here, we established microphysiologic three-dimensional (3D) lung and breast cancer models that resemble architectural and phenotypical features of primary tumors, and evaluated the anti-tumor function of ROR1-specific CAR T-cells. 3D tumors were established from A549 (non-small cell lung cancer) and MDA-MB-231 (triple-negative breast cancer) cell lines on a biological scaffold with intact basement membrane (BM) under static and dynamic culture conditions, which resulted in progressively increasing cell mass and invasive growth phenotype (dynamic>static; MDA-MB-231>A549). Treatment with ROR1-CAR T-cells conferred potent anti-tumor effects. In dynamic culture, CAR T-cells actively entered arterial medium flow, adhered to and infiltrated the tumor mass. ROR1-CAR T-cells penetrated deep into tumor tissue and eliminated multiple layers of tumor cells located above and below the BM. The microphysiologic 3D tumor models developed in this study are standardized scalable test systems that can be used either in conjunction with or in lieu of animal testing to interrogate the anti-tumor function of CAR T-cells, and to obtain proof-of-concept for their safety and efficacy prior to clinical application.
Lars Wallstabe, Claudia Göttlich, Lena C. Nelke, Johanna Kühnemundt, Thomas Schwarz, Thomas Nerreter, Hermann Einsele, Heike Walles, Gudrun Dandekar, Sarah L. Nietzer, Michael Hudecek
Circulating Tumor Cells (CTCs) represent an easy, repeatable and representative access to information regarding solid tumors. However, their detection remains difficult because of their paucity, their short half-life, and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast, sensitive and affordable technique, ideal for rare cells detection. Adapted to CTCs detection (i.e. extremely rare cells), most FC-based techniques require a time-consuming pre-enrichment step, followed by a 2-hours staining procedure, impeding on the efficiency of CTCs detection. We overcame these caveats and reduced the procedure to less than one hour, with minimal manipulation. First, cells were simultaneously fixed, permeabilized, then stained. Second, using low-speed FC acquisition conditions and two discriminators (cell size and pan-cytokeratin expression), we suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases, this protocol ensures a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and can predict which samples are derived from cancer donors. This proof-of-concept study lays the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses.
Alexia Lopresti, Fabrice Malergue, François Bertucci, Maria Lucia Liberatoscioli, Severine Garnier, Quentin DaCosta, Pascal Finetti, Marine Gilabert, Jean Luc Raoul, Daniel Birnbaum, Claire Acquaviva, Emilie Mamessier
Atherosclerotic plaques feature local proliferation of leukocytes and vascular smooth muscle cells (VSMCs) and changes in cellular metabolism. Yet the relationship between glucose utilization and proliferation has been technically impossible to study directly in cells of atherosclerotic plaques in vivo. We used multi-isotope imaging mass spectrometry (MIMS), a quantitative imaging platform, to measure coincident cell division and glucose utilization at suborganelle resolution in atherosclerotic plaques. In established plaques, 65% of intimal foam cells and only 4% of medial VSMCs were labeled with 15N-thymidine after 1 week of isotope treatment. Dividing cells demonstrated heightened glucose labeling. MIMS detected 2H-glucose label in multiple subcellular compartments within foam cells, including lipid droplets, the cytosol, and chromatin. Unexpectedly, we identified an intensely focal region of 2H-label in VSMCs underlying plaques. This signal diminished in regions of aorta without atherosclerosis. In advanced plaques, 15N-thymidine and 2H-glucose labeling in foam cells and VSMCs significantly decreased. These data demonstrate marked heterogeneity in VSMC glucose metabolism that was dependent on both proliferative status and proximity of VSMCs to plaques. Furthermore, these results reveal how quantitative mass spectrometry coupled with isotope imaging can complement other methods used to study cell biology directly in the growing atherosclerotic plaque in vivo.
Christelle Guillermier, Sean P. Doherty, Adam G. Whitney, Vladimir R. Babaev, MacRae F. Linton, Matthew L. Steinhauser, Jonathan D. Brown
The adoptive cell transfer (ACT) of T cells targeting mutated neoantigens can cause objective responses in varieties of metastatic cancers, but the development of new T cell–based treatments relies on accurate animal models. To investigate the therapeutic effect of targeting a neoantigen with ACT, we used T cells from pmel-1 T cell receptor–transgenic mice, known to recognize a WT peptide, gp100, and a mutated version of the peptide that has higher avidity. We gene-engineered B16 cells to express the WT or mutated gp100 epitopes and found that pmel-1–specific T cells targeting a neoantigen tumor target augmented recognition as measured by IFN-γ production. Neoantigen expression by B16 also enhanced the capacity of pmel-1 T cells to trigger the complete and durable regression of large, established, vascularized tumor and required less lymphodepleting conditioning. Targeting neoantigen uncovered the possibility of using enforced expression of the IL-2Rα chain (CD25) in mutation-reactive CD8+ T cells to improve their antitumor functionality. These data reveal that targeting of “mutated-self” neoantigens may lead to improved efficacy and reduced toxicities of T cell–based cellular immunotherapies for patients with cancer.
Ken-ichi Hanada, Zhiya Yu, Gabrielle R. Chappell, Adam S. Park, Nicholas P. Restifo
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