scholarly journals In Vivo Quantitative Assessment of Therapeutic Response to Bortezomib Therapy in Disseminated Animal Models of Multiple Myeloma With [18F]FDG and [64Cu]LLP2A PET

2021 ◽  
Author(s):  
Anchal Ghai ◽  
Nikki Fettig ◽  
Francesca Fontana ◽  
John DiPersio ◽  
Mike Rettig ◽  
...  
Keyword(s):  
Therapeutic Response ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Molar Activity ◽  
Quantitative Pet ◽  
Late Antigen ◽  
Green Fluorescent

Abstract BackgroundMultiple myeloma (MM) is a disease of cancerous plasma cells. Current treatments have improved the survival rate; however, most MM patients relapse. Imaging based timely determination of therapeutic response is critical for improving outcomes in MM patients. Very late antigen-4 (VLA4) is over expressed in MM cells. Here, we evaluated [18F]FDG and VLA4 targeted [64Cu]LLP2A for quantitative PET imaging in MM models of variable VLA4 expression, and following bortezomib therapy.MethodsIn vitro and ex vivo VLA4 expression was evaluated by flow cytometry. Human MM cells, MM.1S-CG and U266-CG (CG: luciferase and green fluorescent protein), were injected intravenously in NOD-SCID gamma mice. Tumor progression was monitored by bioluminescence imaging (BLI). Treatment group received bortezomib (1mg/kg, twice/week) intraperitoneally. All cohorts (treated, untreated and no-tumor) were longitudinally imaged with [64Cu]LLP2A (2-3 MBq; Molar Activity: 44.14±1.40 MBq/nmol) and [18F]FDG (7.4-8.0 MBq) PET respectively.ResultsFlow cytometry confirmed high expression of CD49d in U266 cells (>99%) and moderate expression in MM.1S cells (~52%). BLI showed decrease in total body flux in treated mice. In MM.1S-CG untreated versus treated mice, [64Cu]LLP2A localized with a significantly higher SUVmean in spine (0.58 versus 0.31) and femur (0.72 versus 0.39) at week 4 post tumor inoculation. In U266-CG treated versus untreated mice, there was a 4-time percent [64Cu]LLP2A increase in spine at week 3. Compared to [64Cu]LLP2A, [18F]FDG PET detected treatment related changes at later time points.Conclusion[64Cu]LLP2A is a promising tracer for in vivo assessment of therapeutic response in disseminated models of MM.

scholarly journals In vivo quantitative assessment of therapeutic response to bortezomib therapy in disseminated animal models of multiple myeloma with [18F]FDG and [64Cu]Cu-LLP2A PET

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anchal Ghai ◽  
Nikki Fettig ◽  
Francesca Fontana ◽  
John DiPersio ◽  
Mike Rettig ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Flow Cytometry ◽  
Therapeutic Response ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Molar Activity ◽  
Quantitative Pet

Abstract Background Multiple myeloma (MM) is a disease of cancerous plasma cells in the bone marrow. Imaging-based timely determination of therapeutic response is critical for improving outcomes in MM patients. Very late antigen-4 (VLA4, CD49d/CD29) is overexpressed in MM cells. Here, we evaluated [18F]FDG and VLA4 targeted [64Cu]Cu-LLP2A for quantitative PET imaging in disseminated MM models of variable VLA4 expression, following bortezomib therapy. Methods In vitro and ex vivo VLA4 expression was evaluated by flow cytometry. Human MM cells, MM.1S-CG and U266-CG (C: luciferase and G: green fluorescent protein), were injected intravenously in NOD-SCID gamma mice. Tumor progression was monitored by bioluminescence imaging (BLI). Treatment group received bortezomib (1 mg/kg, twice/week) intraperitoneally. All cohorts (treated, untreated and no tumor) were longitudinally imaged with [18F]FDG (7.4–8.0 MBq) and [64Cu]Cu-LLP2A (2–3 MBq; Molar Activity: 44.14 ± 1.40 MBq/nmol) PET, respectively. Results Flow cytometry confirmed high expression of CD49d in U266 cells (> 99%) and moderate expression in MM.1S cells (~ 52%). BLI showed decrease in total body flux in treated mice. In MM.1S-CG untreated versus treated mice, [64Cu]Cu-LLP2A localized with a significantly higher SUVmean in spine (0.58 versus 0.31, p < 0.01) and femur (0.72 versus 0.39, p < 0.05) at week 4 post-tumor inoculation. There was a four-fold higher uptake of [64Cu]Cu-LLP2A (SUVmean) in untreated U266-CG mice compared to treated mice at 3 weeks post-treatment. Compared to [64Cu]Cu-LLP2A, [18F]FDG PET detected treatment-related changes at later time points. Conclusion [64Cu]Cu-LLP2A is a promising tracer for timely in vivo assessment of therapeutic response in disseminated models of MM.

scholarly journals Plasma Cell Ontogeny Defined by Quantitative Changes in Blimp-1 Expression

2004 ◽  
Vol 200 (8) ◽  
pp. 967-977 ◽  
Author(s):  
Axel Kallies ◽  
Jhagvaral Hasbold ◽  
David M. Tarlinton ◽  
Wendy Dietrich ◽  
Lynn M. Corcoran ◽  
...  
Keyword(s):  
Plasma Cell ◽  
B Lymphocyte ◽  
Plasma Cells ◽  
Fluorescent Protein ◽  
Developmental Stages ◽  
Maturation Protein ◽  
Quantitative Changes ◽  
Green Fluorescent

Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte–induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression.

scholarly journals Electrosprayed Alginate Nanoparticles as CRISPR Plasmid DNA Delivery Carrier: Preparation, Optimization, and Characterization

2020 ◽  
Vol 13 (8) ◽  
pp. 158 ◽  
Author(s):  
Batoul Alallam ◽  
Sara Altahhan ◽  
Muhammad Taher ◽  
Mohd Hamzah Mohd Nasir ◽  
Abd Almonem Doolaanea
Keyword(s):  
Fluorescent Protein ◽  
Biomedical Application ◽  
Ex Vivo ◽  
Strand Break ◽  
Successful Implementation ◽  
Mean Size ◽  
Green Fluorescent ◽  
In Vivo Delivery

Therapeutic gene editing is becoming more feasible with the emergence of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) system. However, the successful implementation of CRISPR/Cas9-based therapeutics requires a safe and efficient in vivo delivery of the CRISPR components, which remains challenging. This study presents successful preparation, optimization, and characterization of alginate nanoparticles (ALG NPs), loaded with two CRISPR plasmids, using electrospray technique. The aim of this delivery system is to edit a target gene in another plasmid (green fluorescent protein (GFP)). The effect of formulation and process variables were evaluated. CRISPR ALG NPs showed mean size and zeta potential of 228 nm and −4.42 mV, respectively. Over 99.0% encapsulation efficiency was achieved while preserving payload integrity. The presence of CRISPR plasmids in the ALG NPs was confirmed by Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy. The tests revealed that the nanoparticles were cytocompatible and successfully introduced the Cas9 transgene in HepG2 cells. Nanoparticles-transfected HepG2 was able to edit its target plasmid by introducing double-strand break (DSB) in GFP gene, indicating the bioactivity of CRISPR plasmids encapsulated in alginate nanoparticles. This suggests that this method is suitable for biomedical application in vitro or ex vivo. Future investigation of theses nanoparticles might result in nanocarrier suitable for in vivo delivery of CRISPR/Cas9 system.

scholarly journals Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

2021 ◽  
Vol 9 (2) ◽  
pp. 379
Author(s):  
Breanne M. Head ◽  
Christopher I. Graham ◽  
Teassa MacMartin ◽  
Yoav Keynan ◽  
Ann Karen C. Brassinga
Keyword(s):  
Growth Kinetics ◽  
Legionella Pneumophila ◽  
Fluorescent Protein ◽  
Disease Incidence ◽  
Acanthamoeba Castellanii ◽  
Infection Model ◽  
Intracellular Infection ◽  
Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

scholarly journals CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
Peng-Fei Fu ◽  
Xuan Cheng ◽  
Bing-Qian Su ◽  
Li-Fang Duan ◽  
Cong-Rong Wang ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Antiviral Agents ◽  
Firefly Luciferase ◽  
Inhibitory Effect ◽  
Economic Losses ◽  
Green Fluorescent ◽  
Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

scholarly journals The Great Capacity on Promoting Melanogenesis of Three Compatible Components in Vernonia anthelmintica (L.) Willd.

2021 ◽  
Vol 22 (8) ◽  
pp. 4073
Author(s):  
Yifan Lai ◽  
Qingyuan Feng ◽  
Rui Zhang ◽  
Jing Shang ◽  
Hui Zhong
Keyword(s):  
Herbal Medicine ◽  
Caffeic Acid ◽  
Fluorescent Protein ◽  
Traditional Chinese Herbal Medicine ◽  
Expression Of Genes ◽  
Proliferation And Differentiation ◽  
Green Fluorescent ◽  
Vernonia Anthelmintica

To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of skin depigmentation disorder, we have made use of Vernonia anthelmintica (L.) Willd., a traditional Chinese herbal medicine that has been proven to be effective in treating vitiligo. Here, we report that the extract of Vernonia anthelmintica (L.) Willd. effectively enhances melanogenesis responses in B16F10. In its compound library, we found three ingredients (butin, caffeic acid and luteolin) also have the activity of promoting melanogenesis in vivo and in vitro. They can reduce the accumulation of ROS induced by hydrogen peroxide and inflammatory response induced by sublethal concentrations of copper sulfate in wild type and green fluorescent protein (GFP)-labeled leukocytes zebrafish larvae. The overall objective of the present study aims to identify which compatibility proportions of the medicines may be more effective in promoting pigmentation. We utilized the D-optimal response surface methodology to optimize the ratio among three molecules. Combining three indicators of promoting melanogenesis, anti-inflammatory and antioxidant capacities, we get the best effect of butin, caffeic acid and luteolin at the ratio (butin:caffeic acid:luteolin = 7.38:28.30:64.32) on zebrafish. Moreover, the effect of melanin content recovery in the best combination is stronger than that of the monomer, which suggests that the three compounds have a synergistic effect on inducing melanogenesis. After simply verifying the result, we performed in situ hybridization on whole-mount zebrafish embryos to further explore the effects of multi-drugs combination on the proliferation and differentiation of melanocytes and the expression of genes (tyr, mitfa, dct, kit) related to melanin synthesis. In conclusion, the above three compatible compounds can significantly enhance melanogenesis and improve depigmentation in vivo.

scholarly journals Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

2021 ◽  
Vol 30 ◽  
pp. 096368972097821
Author(s):  
Andrea Tenorio-Mina ◽  
Daniel Cortés ◽  
Joel Esquivel-Estudillo ◽  
Adolfo López-Ornelas ◽  
Alejandro Cabrera-Wrooman ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Ectopic Expression ◽  
Neural Induction ◽  
Human Keratinocytes ◽  
Differentiation Potential ◽  
Neuronal Proteins ◽  
Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

Atelocollagen Sponge as a Stem Cell Implantation Scaffold for the Treatment of Scarred Vocal Folds

2010 ◽  
Vol 119 (11) ◽  
pp. 805-810 ◽  
Author(s):  
Satoshi Ohno ◽  
Shigeru Hirano ◽  
Ichiro Tateya ◽  
Shin-Ichi Kanemaru ◽  
Hiroo Umeda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Stromal Cells ◽  
Fluorescent Protein ◽  
In Vitro Study ◽  
Vocal Folds ◽  
Green Fluorescent ◽  
Cell Implantation

Objectives: Treatment of vocal fold scarring remains a therapeutic challenge. Our group previously reported the efficacy of treating injured vocal folds by implantation of bone marrow—derived stromal cells containing mesenchymal stem cells. Appropriate scaffolding is necessary for the stem cell implant to achieve optimal results. Terudermis is an atelocollagen sponge derived from calf dermis. It has large pores that permit cellular entry and is degraded in vivo. These characteristics suggest that this material may be a good candidate for use as scaffolding for implantation of cells. The present in vitro study investigated the feasibility of using Terudermis as such a scaffold. Methods: Bone marrow—derived stromal cells were obtained from GFP (green fluorescent protein) mouse femurs. The cells were seeded into Terudermis and incubated for 5 days. Their survival, proliferation, and expression of extracellular matrix were examined. Results: Bone marrow—derived stromal cells adhered to Terudermis and underwent significant proliferation. Immunohistochemical examination demonstrated that adherent cells were positive for expression of vimentin, desmin, fibronectin, and fsp1 and negative for beta III tubulin. These findings indicate that these cells were mesodermal cells and attached to the atelocollagen fibers biologically. Conclusions: The data suggest that Terudermis may have potential as stem cell implantation scaffolding for the treatment of scarred vocal folds.

In vitro and in vivo evaluation of a green fluorescent protein-HSV1-TK dual-function pet reporter gene

2001 ◽  
Vol 44 (S1) ◽  
pp. S339-S341
Author(s):  
K. E. Luker ◽  
G. D. Luker ◽  
C. M. Pica ◽  
J. L. Dahlheimer ◽  
T. J. Fahrner ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Reporter Gene ◽  
Fluorescent Protein ◽  
Dual Function ◽  
In Vivo Evaluation ◽  
Green Fluorescent

scholarly journals A novel BCMA/CD3 bispecific T-cell engager for the treatment of multiple myeloma induces selective lysis in vitro and in vivo

Leukemia ◽  
2016 ◽  
Vol 31 (8) ◽  
pp. 1743-1751 ◽  
Author(s):  
S Hipp ◽  
Y-T Tai ◽  
D Blanset ◽  
P Deegen ◽  
J Wahl ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
T Cell ◽  
Plasma Cells ◽  
Ex Vivo ◽  
Xenograft Model ◽  
Selective Lysis ◽  
Bispecific T Cell Engager

Abstract B-cell maturation antigen (BCMA) is a highly plasma cell-selective protein that is expressed on malignant plasma cells of multiple myeloma (MM) patients and therefore is an ideal target for T-cell redirecting therapies. We developed a bispecific T-cell engager (BiTE) targeting BCMA and CD3ɛ (BI 836909) and studied its therapeutic impacts on MM. BI 836909 induced selective lysis of BCMA-positive MM cells, activation of T cells, release of cytokines and T-cell proliferation; whereas BCMA-negative cells were not affected. Activity of BI 836909 was not influenced by the presence of bone marrow stromal cells, soluble BCMA or a proliferation-inducing ligand (APRIL). In ex vivo assays, BI 836909 induced potent autologous MM cell lysis in both, newly diagnosed and relapsed/refractory patient samples. In mouse xenograft studies, BI 836909 induced tumor cell depletion in a subcutaneous NCI-H929 xenograft model and prolonged survival in an orthotopic L-363 xenograft model. In a cynomolgus monkey study, administration of BI 836909 led to depletion of BCMA-positive plasma cells in the bone marrow. Taken together, these results show that BI 836909 is a highly potent and efficacious approach to selectively deplete BCMA-positive MM cells and represents a novel immunotherapeutic for the treatment of MM.

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