scholarly journals In Vivo Inhibition of MicroRNA-326 in a NOD.H-2h4 Mouse Model of Autoimmune Thyroiditis

2021 ◽  
Vol 12 ◽  
Author(s):  
Na Zhao ◽  
Zhenzhen Wang ◽  
Xuejiao Cui ◽  
Shuo Wang ◽  
Chenling Fan ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Autoimmune Thyroiditis ◽  
Lentiviral Vector ◽  
Control Group ◽  
Tail Vein ◽  
Inflammatory Score ◽  
Tail Vein Injection ◽  
Hematoxylin And Eosin ◽  
Inhibition Group

BackgroundPrevious studies reported that various miRNAs participate in autoimmune diseases, but the potential regulatory mechanism of miRNAs in autoimmune thyroiditis (AIT) needs further exploration.ObjectiveThis study aimed to further verify that miR-326 contributes to AIT by regulating Th17/Treg balance through Ets-1 using lentiviral gene delivery through tail vein and thyroid injection in NOD.H-2h4 mice.Materials and MethodsFive-week-old NOD.H-2h4 mice were divided randomly into tail vein and thyroid injection groups, and each received either mmu-miR-326 sponge (LV-sponge) or lentiviral vector control. Mice were divided for tail vein injection: the therapeutic LV-ctrl, therapeutic LV-sponge, prophylactic LV-ctrl, and prophylactic LV-sponge groups. The control group was fed high-iodine water without vein injection. The thyroid infiltration of lymphocytes and serum TgAb value were investigated by thyroid hematoxylin and eosin (HE) staining and ELISA, respectively. Ets-1 and lymphocyte counts were measured by RT-PCR, western blotting, and flow cytometry. The thyroid CD4+IL-17a+ cells and CD4+Ets-1+ cells were detected by immunofluorescence, and the serum cytokines were tested by ELISA.ResultsIn the tail vein injection groups, the thyroid inflammatory score and serum TgAb titer were significantly lower in the LV-sponge groups than in the control and LV-ctrl groups while Ets-1 protein expression in mouse spleens was increased in the LV-sponge groups. Moreover, Th17/Treg ratio declined in the LV-sponge group and decreased significantly in the prophylactic LV-sponge group (P = 0.036) tested by flow cytometry. Immunofluorescence showed that, in LV-sponge groups, CD4+IL-17a+ cells were decreased significantly (P = 0.001), while CD4+Ets-1+ cells were increased significantly in the LV-sponge group (P = 0.029). The serum IL-17/IL-10 was decreased significantly in the LV-sponge group (P < 0.05). In the thyroid injection groups, the thyroid inflammatory score and serum TgAb titer in the LV-sponge group decreased significantly compared with those in the LV-ctrl group (P < 0.05). In addition, in LV-sponge groups, CD4+IL-17a+ cells were decreased, while CD4+Ets-1+ cells were increased significantly in the inhibition group evaluated by immunofluorescence. Moreover, tail vein injection of LV-sponge resulted in much lower TgAb levels in thyroiditis compared with thyroid injection.ConclusionMiR-326 targeted therapy may be a promising approach for AIT. In addition, tail vein injection may achieve a better intervention effect than thyroid injection.

scholarly journals Characterization of human follicular thyroid cancer cell lines in preclinical mouse models

2016 ◽  
Vol 5 (2) ◽  
pp. 47-54 ◽  
Author(s):  
Ashley N Reeb ◽  
Andrea Ziegler ◽  
Reigh-Yi Lin
Keyword(s):  
Thyroid Cancer ◽  
Cell Lines ◽  
Lung Metastasis ◽  
Lung Metastases ◽  
Metastatic Potential ◽  
Follicular Thyroid Cancer ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Injection Model

Follicular thyroid cancer (FTC) is the second most common type of thyroid cancers. In order to develop more effective personalized therapies, it is necessary to thoroughly evaluate patient-derived cell lines in in vivo preclinical models before using them to test new, targeted therapies. This study evaluates the tumorigenic and metastatic potential of a panel of three human FTC cell lines (WRO, FTC-238, and TT1609-CO2) with defined genetic mutations in two in vivo murine models: an orthotopic thyroid cancer model to study tumor progression and a tail vein injection model to study metastasis. All cell lines developed tumors in the orthotopic model, with take rates of 100%. Notably, WRO-derived tumors grew two to four times faster than tumors arising from the FTC-238 and TT2609-CO2 cell lines. These results mirrored those of a tail vein injection model for lung metastasis: one hundred percent of mice injected with WRO cells in the tail vein exhibited aggressive growth of bilateral lung metastases within 35 days. In contrast, tail vein injection of FTC-238 or TT2609-CO2 cells did not result in lung metastasis. Together, our work demonstrates that these human FTC cell lines display highly varied tumorigenic and metastatic potential in vivo with WRO being the most aggressive cell line in both orthotopic and lung metastasis models. This information will be valuable when selecting cell lines for preclinical drug testing.

scholarly journals Rara Is a Druggable Super-Enhancer Regulated Dependency in Pediatric AML

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 281-281
Author(s):  
Alfred S. Daramola ◽  
Monika Perez ◽  
Oscar S. Sias-Garcia ◽  
Maci S. Terrell ◽  
Helen Y Wei ◽  
...  
Keyword(s):  
Transcriptional Regulation ◽  
Retinoic Acid ◽  
Cell Lines ◽  
Phase Ii ◽  
Mrna Levels ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Gene And Protein Expression ◽  
Pediatric Aml

Introduction: New approaches to find and then drug pediatric acute myeloid leukemia (AML)-specific targets are clearly needed to help the nearly 35% of patients who still die from the disease. While RARA is a known druggable target in acute promyelocytic leukemia (APL), the utility of using retinoic acid agonists in non-APL AML has not proven consistently beneficial. Super enhancers (SEs), large regions of highly active chromatin, define cell state and cell identity by regulating oncogenes in many cancers. Recent enhancer profiling of 66 adult non-APL AML patient samples revealed SE-defined, prognostically relevant subgroups. An SE was detected at the retinoic acid receptor alpha (RARA) gene locus in 59% of the samples, which were sensitive to the second-generation retinoic acid agonist tamibarotene which has led to a phase II clinical trial. This study confirms that characterization of the chromatin-defined dependencies in specific cancers can pinpoint targets which can be drugged. We are delineating the transcriptional regulation of pediatric AML (pAML) by SE analysis, which has already elucidated deeper insights into pediatric leukemogenesis, as typified by strong RARA dependence in a majority of pAML. Methods: Three AML cell lines and 19 pAML primary samples were enhancer profiled by H3K27Ac chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq). SEs were detected and assigned to genes using the rank ordering of super-enhancers (ROSE) algorithm. Tamibarotene treatment of cell lines and patient samples were assessed for gene and protein expression changes and phenotypic differences. For in vivo assessment, 200,000 cells of a RARA SE+ pAML patient sample were injected into each NSGS mouse by tail-vein injection. One week after injection, tamibarotene (6mg/kg) or vehicle treatment was initiated by gavage (n=7 each arm). Peripheral blood monitoring of leukemia burden was determined flow cytometry. Results: The primary pAML sample cohort encompassed the diverse pAML cytogenetic subtypes (Fig 1a), with an overrepresentation of KMT2A rearrangements (n=9, 47%). The number of unique enhancer regions was nearly saturated in the 19 samples. Median SE size was 3,780bp, much larger than the 511bp of typical enhancers. When SE regions across all samples were clustered together, a RARA SE was seen in two of the ten clusters (Fig 1b). Eleven of the 19 samples (58%) contained a RARA SE, crossing multiple cytogenetic subtypes (Fig 1c). Tamibarotene treatment of RARA SE+ pAML cell lines and patient samples suppressed proliferation and increased apoptosis (detected by annexin V+), with minimal effect in Kasumi, a pAML cell line without a RARA SE (Fig 2a). In the RARA SE+ cell lines and samples only, tamibarotene increased CD38 (a myeloid differentiation marker usually suppressed by ligand-unbound RARA) (Fig 2b). High RARA mRNA levels confirmed the SE assignments in both cell lines and patient samples (Fig 2c). Tamibarotene induced the transcription of DHRS3 (another RARA target gene used as a pharmacodynamic biomarker in the adult tamibarotene phase II trial) (Fig 2d). Tamibarotene suppressed colony formation ability in RARA SE+ cell lines. An ongoing RARA SE+ patient-derived pAML xenograft confirmed tamibarotene markedly suppressed disease progression (Fig 3), with vehicle mice requiring euthanasia 42 days after tail-vein injection for significant disease burden, while the tamibarotene treated mice continued to be well-appearing at the same timepoint. Conclusion: We have profiled the enhancer landscapes of 19 primary pAML samples, the largest dataset of its kind, and seen a high frequency of a RARA SE in pAML. Tamibarotene has anti-proliferative, proapoptotic, and on-target pro-differentiation effects in RARA SE+ pAML in vitro and marked anti-leukemia activity in vivo. Given these positive findings, we are evaluating combinations of other AML-active agents with tamibarotene. Additionally, as there is a range of sensitivity to tamibarotene in RARA SE+ samples, we are also interrogating other SE-regulated genes interacting with RARA that may predict degree of response or resistance to tamibarotene. Our studies confirm that studying the transcriptional regulation of pAML samples through SE analysis can identify druggable targets and also lay the preclinical foundation for a biomarker-defined tamibarotene trial in pediatric AML. Disclosures Wei: NHI NHLBI Grant: Other: received funding . Lin:Syros Pharmaceuticals: Equity Ownership, Patents & Royalties.

A SCID Mouse Model for Monitoring in Vivo Viability of Human Platelet Concentrates Using Flow Cytometry

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4549-4549
Author(s):  
Jia-hua Ding ◽  
Cheng-yin Huang ◽  
Shiyun Xu
Keyword(s):  
Flow Cytometry ◽  
Whole Blood ◽  
Human Platelet ◽  
Human Platelets ◽  
Scid Mice ◽  
Test Method ◽  
Platelet Concentrates ◽  
Tail Vein

Abstract Objective To develop an animal test method for evaluating the in vivo quality of human platelet concentrates. Methods Human platelets were transfused to mice by tail vein with a 1mL insulin syring fitted with a 29-gauge ultra-fine needle. Blood samples were taken at 30 minutes,2,4,6,8,12, and 24hours after infusion with a tail vein nick technique, whole blood was collected into heparinized capillary tubes. Human platelets in mouse whole blood were detected by flow cytometry with monoclonal anti-human CD61-PE–conjugated antibodies. All subsequent recoveries were calculated as a percentage of the initial collection. Results The survival time of human platelets were significantly prolonged in SCID than in BALB/c,FVB mice. Recoveries at 4 hours after transfusion in SCID, BALB/c,FVB mice were 68.6%±8.1%(n =10),29.9%±6.5%(n =8),28.1%±5.5%(n =8), respectively, and with a T½ estimate of 8 hours for SCID, 2.5 hours for BALB/c and 2 hours for FVB mice. platelet storage lesions either by chemical treatment or by suboptimal conditions storage exhibited decreased recoveries in SCID mice. Conclusion The quality of platelet Products can be evaluated by assessing the survival of human platelets in SCID mice using flow cytometry.

scholarly journals High-resolution hyperpolarized in vivo metabolic 13C spectroscopy at low magnetic field (48.7 mT) following murine tail-vein injection

2017 ◽  
Vol 281 ◽  
pp. 246-252 ◽  
Author(s):  
Aaron M. Coffey ◽  
Matthew A. Feldman ◽  
Roman V. Shchepin ◽  
Danila A. Barskiy ◽  
Milton L. Truong ◽  
...  
Keyword(s):  
Magnetic Field ◽  
High Resolution ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Low Magnetic Field

Feasibility study of morphine-induced rat pica model by tail vein injection to replace vomiting model

2020 ◽  
Vol 20 (5) ◽  
pp. 364-366
Author(s):  
Weijian ZHOU ◽  
◽  
Pengju BAO ◽  
Wenjun GUO ◽  
Ting HONG ◽  
...  
Keyword(s):  
Oxygen Saturation ◽  
Effective Dose ◽  
Statistical Significance ◽  
Control Group ◽  
Blood Oxygen ◽  
Blank Control Group ◽  
Blood Oxygen Saturation ◽  
Treatment Groups ◽  
Tail Vein ◽  
Tail Vein Injection

Objective： To explore the feasibility of the morphine-induced rat pica model by tail vein injection to replace the vomiting model，and the effective dose of morphine with less severe complications when the model was developed.Methods： Fifty SD rats were randomly divided into the blank control group and the M1，M2，M3 and M4 groups，each consisting of 10 animals. The rats in the blank control group were injected with 10 ml/kg normal saline（NS） via tail vein，while the animals in the M1，M2，M3 and M4 groups were injected with morphine hydrochloride injection at a dosage of 5，10，20 and 30 mg/kg via tail vein.Heart rate，blood oxygen saturation and activities of the animals were closely observed and recorded at 5，10 and 15 minutes after medication.Then，changes in the intake of kaolin，feed and water of the rats in each group were recorded and compared between the groups for 4 successive days after medication.Results： The blood oxygen saturation in the morphine treatment groups all reduced significantly，and statistical significance could be seen，when it was compared with that of the blank control group（P<0.05）.On the first day after medication，the intake of kaolin in the M3 and M4 groups increased significantly，as compared with that of the blank control group（P<0.05）.On the third day after medication，the intake of kaolin in the M1 group significantly increased，as compared with that of the blank control group（P<0.05）.Conclusion： The rat pica model was established by tail vein injection of morphine，which could be used to replace the vomiting model.The effective dose of morphine was 20 mg/kg with less severe complications.

Tumor Homing Activity of Bone Marrow-Derived Mesenchymal Stem Cell Is Highly Various Among Different Tumors On Syngeneic Mouse Model Using Real-Time in Vivo Imaging Technique.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1453-1453
Author(s):  
Su-Peng Yeh ◽  
Wen-Jyi Lo ◽  
Yu-Chien Chang ◽  
Wan-Ju Tsai ◽  
Chiao-Lin Lin ◽  
...  
Keyword(s):  
Cell Line ◽  
Cancer Cell ◽  
Luciferase Activity ◽  
Cancer Cell Line ◽  
Firefly Luciferase ◽  
Tail Vein ◽  
Tail Vein Injection ◽  
Syngeneic Mouse Model ◽  
Tumor Types

Abstract Abstract 1453 Poster Board I-476 Purpose Mesenchymal stem cells (MSCs) have the unique ability of homing to tumor tissue. Most of prior studies using GPF-labeled MSCs in the xenograft models (hMSC-GFP homing to human cancer on the immunodeficient mice) and then counting the GFP(+) cells in the tumor and other tissues to evaluate homing specificity. This model is far away from clinically relevant condition and bios would develop during counting process. More importantly, it remains unknown whether MSCs target specific or all the tumor types in immunocompotent host. Here we use firefly luciferase stably expressed MSCs cell line (D1-Luc) derived from bone marrow of balb/c mice and assess the homing activity on different tumor types (also derived from balb/c mice) by using in vivo imaging system (IVIS). Materials & Methods D1 cells were purchased from ATCC. Firefly luciferase (Luc) stably expressed D1 was selected and maintained after transducing Luc-Neomycin into D1. 4T1, CT26, and Rag cells (breast cancer cell line, colon cancer cell line, and renal cancer cell line derived from balb/c mice) was subcutaneously injected into 6 to 8 weeks balb/c mice and tumor would develop within 1 week. 2 × 106 D1-luc cells were then injected into normal balb/c or tumor-bearing balb/c through tail vein. 1 hour, 1 day, 3 days, 7 days and 14 days after tail vein injection, balb/c mice were anesthetized and luciferase activity in the whole mouse was evaluated by IVIS (xenogen). Results 1 hour after D1-luc tail vein injection, luciferase activity was detected mostly in the lung (normal balb/c and CT26-babl/c) and both lung and tumor (4T1-balb/c and Rag-balb/c). The luciferase activity in lung decreased markedly 1 day after injection and no longer detectable after 3 days in both normal and tumor-bearing mice. However, the luciferase activity accumulated in tumor tissue markedly and progressively increased in 4T1-balb/c and Rag-balb/c since 1 day after tail vein injection and persisted for more than 1 week. The luciferase activity in lung was detectable again 2 weeks after tail vein injection in few 4T1- and Rag-bearing mice. Lung tissue was examined and tumor metastasis in lung was confirmed in these mice. Furthermore, luciferase-positive cells can be detected in the metastatic tumor nests by using immunohistochemical staining. Luciferase activity was not detected at tumor sites of CT26-balb/c at any time point after injection of D1-Luc. Conclusions In this immunocompotent, syngeneic mouse model, BM-MSCs showed a highly various tumor-homing activity. BM-MSCs home to tumor site of breast (4T1) and renal cancer (Rag)-bearing balb/c mice quickly and specifically. By contrary, BM-MSCs homing to tumor site of colon cancer (CT26)-bearing mice was below the detectable limit of IVIS. Our findings suggest MSCs homing to tumor is not a universal conclusion and there must be some mechanism driving MSCs homing to a specific type of tumor rather than all the tumors. However, for a tumor that MSCs specifically targeted, MSCs can home to the primary tumor and also the metastatic site. This information is very important when considering MSCs as a vehicle of cell or gene therapy for cancer. Disclosures No relevant conflicts of interest to declare.

scholarly journals Melittin Inducing the Apoptosis of Renal Tubule Epithelial Cells through Upregulation of Bax/Bcl-2 Expression and Activation of TNF-α Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-13 ◽  
Author(s):  
Ying Shu ◽  
Yingying Yang ◽  
Yuliang Zhao ◽  
Liang Ma ◽  
Ping Fu ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Signaling Pathway ◽  
Kidney Injury ◽  
Protein Detection ◽  
Severe Illness ◽  
Control Group ◽  
Mrna Levels ◽  
Tail Vein

Background. Acute kidney injury (AKI) caused by bee stings is common, with characteristics of acute onset, severe illness, and high mortality. Melittin, a major component of bee venom, has been considered to play a key role in bee sting related AKI. This study aims to illustrate whether melittin could lead to apoptosis of renal tubular epithelial cells (RTECs) and to investigate its mechanism. Methods. In vivo, 45 mice were randomly divided into the melittin group (n=30, injected with melittin into the tail vein according to the total dose of 4.0 ug/g weight) and the control group (n=15, injected with the same volume of saline into the tail vein). In vitro, human RTECs (HK-2) were cultured and treated with melittin (2ug/ml or 4ug/ml) and TNF-α (10ng/ml). Biochemical analysis, HE stains, and electron microscope were performed to evaluate renal function and pathological changes. TUNEL stains and flow cytometry were performed to detect apoptosis. Real-time PCR was performed to detect mRNA levels of Bax, Bcl-2, and TNF-α. Simple western assay and immunohistochemical (IH) and immunofluorescent (IF) stains were performed for protein detection. Results. Melittin successfully induced AKI in mice. Compared with the control group, obvious injury and apoptosis of RTECs were observed in the melittin group; the mRNA and protein expressions of Bax were significantly increased, while the expression of Bcl-2 was significantly decreased. The serum TNF-αlevel in melittin group was significantly higher than that in control group. In vitro, the results confirmed that melittin can cause HK-2 cells apoptosis. The trends of expression of Bax and Bcl-2 were consistent with the results in vivo. The levels of TNF-α mRNA and protein by PCR and Western blot were significantly higher in melittin group than those in control group. Conclusion. Melittin can lead to the apoptosis of RTECs, which may be mediated by upregulating the expression of Bax/Bcl-2 and activating the TNF-α signaling pathway.

scholarly journals Percutaneous ultrasound guided PEG-coated gold nanoparticles enhanced radiofrequency ablation in liver

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tudor Mocan ◽  
Rares Stiufiuc ◽  
Calin Popa ◽  
Iuliana Nenu ◽  
Cosmin Pestean ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Radiofrequency Ablation ◽  
Physiological Saline ◽  
Control Group ◽  
Target Tissue ◽  
Porcine Liver ◽  
Contrast Enhanced ◽  
Hematoxylin And Eosin ◽  
Coagulation Zone

AbstractTo investigate the effects of PEG-coated gold nanoparticles on ablation zone volumes following in vivo radiofrequency ablation of porcine liver. This prospective study was performed following institutional animal care and committee approval was used. Radiofrequency ablations were performed in the livers of ten Sus scrofa domesticus swines. During each ablation, 10 mL (mL) of Peg-coated gold nanoparticles at two different concentrations (0.5 mg/mL and 0.01 mg/mL) were injected through the electrode channel into the target zone. For the control group, 10 mL of physiological saline was used. Five to ten minutes after each ablation, contrast enhanced ultrasound (CEUS) was performed to evaluate the volume of the coagulation zone. On day five we performed another CEUS and the animals were sacrificed. Treated tissues were explanted for quantification of the ablation zones’ volumes. Hematoxylin and eosin (H&E) staining was also performed for histologic analysis. A total of 30 ablations were performed in the livers. The mean coagulation zone volume as measured by CEUS on day 5 after RFA was: 21.69 ± 3.39 cm3, 19.22 ± 5.77 cm3, and 8.80 ± 3.33 cm3 for N1, N2 and PS respectively. The coagulation zone volume after N1 and N2 treatments was significantly higher compared to PS treatment (p < 0.001 and p = 0.025 respectively). There was no difference between N1 and N2 treatment (p = 0.60). In our proof-of concept, pilot study we have shown for the first time that when injected directly into the target tissue during RFA, gold nanoparticles can substantially increase the coagulation zone.

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