Irreversible electroporation (IRE) is a promising technique for the focal treatment of pathologic tissues, which involves placing minimally invasive electrodes within the targeted region. However, the knowledge about the therapeutic efficacy and immune reactions in response to IRE remains in its infancy.In this work, to detect whether tumor ablation with IRE could trigger the immunologic response, we developed an osteosarcoma rat model and applied IRE directly to ablate the tumor. In the experiment, 118 SD rats were randomized into 4 groups: the control, sham operation, surgical resection, and IRE groups. Another 28 rats without tumor cell implantation served as the normal non-tumor-bearing group. We analyzed the changes in T lymphocyte subsets, sIL-2R and IL-10 levels in the peripheral blood one day before operation, as well as at 1, 3, 7,14 and 21 days after the operation. Moreover, splenocytes were assayed for IFN-γ and IL-4 production using intracellular cytokine staining one day before the operation, as well as at 7 and 21 days after operation.We found that direct IRE completely ablated the tumor cells. A significant increase in peripheral lymphocytes, especially CD3(+) and CD4(+) cells, as well as an increased ratio of CD4(+)/CD8(+) were detectable 7 days after operation in both the IRE and surgical resection groups. Compared with the surgical resection group, the IRE group exhibited a stronger cellular immune response. The sIL-2R level of the peripheral blood in the IRE group decreased with time and was significantly different from that in the surgical resection group. Moreover, ablation with IRE significantly increased the percentage of IFN-γ-positive splenocytes.These findings indicated that IRE could not only locally destroy the tumor but also change the status of cellular immunity in osteosarcoma-bearing rats. This provides experimental evidence for the clinical application of IRE in osteosarcoma treatment.
OBJECTIVE The senescence and death of nucleus pulposus (NP) cells are the pathologic basis of intervertebral disc degeneration (IVD). To investigate the molecular phenotypes and senescent mechanism of NP cells, and to identify the method of alleviating senescence of NP cells.The primary NP cells were harvested from male Sprague Dawley rats (8-10 weeks old); the hypoxia inducible factor 1alpha(HIF-1alpha, HIF-1beta matrix metalloproteinase 2 (MMP-2), and collagen type II as phenotypic markers were identified through immunocytochemical staining. RT-PCR and Western blot were used to test the silencing effect of NP cells after the NP cells were transfected with p53 and p21 small interference RNA (siRNA). Senescence associated-beta-galactosidase (SA-beta-gal) staining was used to test the senescence of NP cells, flow cytometry to test the change of cell cycle, the growth curve analysis to test the NP cells proliferation.Immunocytochemical staining showed that NP cells expressed HIF-1alpha HIF-1beta, MMP-2, and collagen type II. RT-PCR and Western blot showed that the relative expressions of mRNA and protein of p53 and p21 were significantly inhibited in NP cells at passage 35 after transfected with p53 and p21 siRNA. The percentage of SA-Pbetagal-positive NP cells at passage 35 was significantly higher than that at passage 1 (P < 0.001). And the percentage of SA-Pbetagal-positive NP cells in the p53 siRNA transfection group and p21 siRNA transfection group were significantly lower than that in control group (P < 0.001). The flow cytometry showed that the GC1phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group was significantly shorter than that in control group (P < 0.05), but the S phase of NP cells in p53 siRNA transfection group and p21 siRNA transfection group were significantly longer than that in control group (P < 0.05). In addition, the growth curve showed that the growth rate of NP cells could be promoted after transfection of p53 and p21 siRNA.he senescence of NP cells can be alleviated by silencing of p53 and p21. The effect of alleviating senescence can even ameliorate the progress of IVD and may be a useful and potential therapy for IVD.
Background/Purpose:
One and a half million Americans suffer from systemic lupus erythematosus (SLE), with increased death rate in the 15–44 year age group. SLE has a wide range of symptoms and affects multiple organs and tissues. SLE affects each person differently over time. Treatment options are limited with potential serious side effects. There is no cure currently due to the lack of understanding of the mechanism of disease at molecular levels. However, recent developments in the field of molecular imaging provide a tool for noninvasive, target-specific and longitudinal study of multiple disease components at the same time as well as understanding the dynamic change of the disease. We hypothesize genetic differences play a role in development and severity of the disease. In addition, multiple different disease molecules in different organs at one time point make SLE difficult to treat by standard regiments.
Methods:
We tested our hypothesis in animal models with two different genetic backgrounds and three target-specific molecular imaging agents. Two groups of mice carrying normal or mutant Fas gene with SLE were utilized in the study. Optical imaging agents that specifically target to pro-inflammatory cytokine (IL-11), cytokine from T-cells (CXCR4), or disease stroma (matrix metalloproteinase, MMP) were injected through the tail vein. Mice were imaged immediately after injection and for as long as 48 hours afterward. SAS software was used to analyze data by one-way-ANOVA or the general linear model. Data comparison was presented in notched box-and whisker plots. The significant level was set at 0.05 with two-tailed test.
Results:
Mice with a normal Fas gene have statistical higher signal intensities for all agents in the joint regions than the Fas mutant mice (P = 0.0139). Target-specific agents have different organ distributions in Fas mutant mice. The MMP signals are in the kidney (Fig. 1A) and IL-11 signals are in the lung (Fig. 1B). Both agents have statistical low signal intensities in the same organs in the control mice with normal Fas gene (P<0.0002). The animal with normal Fas gene has high IL-11 signal intensity in the knee (Fig. 1C). On contrary, CXCR4 has significantly low signal intensity in the Fas mutant mice. Fas mutant mice also have enlarged spleen and kidney by either size or weight than control mice (P<0.0001).
Conclusion:
We found that different genetic backgrounds predispose to different disease manifestations. Each organ has different disease components even with same the genetic background at the same disease stage The method we developed here can be used as a tool to better understand genetic and disease components and improve the treatment plan for individual patient needs.
Aim To identify the specificity of monoclonal antibody(mAb) 5D3 against human osteosarcoma. Methods The reactivities of mAb 5D3 to osteosarcoma tissues, other tumor tissues and more than 14 kinds of normal tissues were detected by enzyme-labeled immunohistochemical staining. Results The mAb 5D3-associated antigen was highly expressed in human osteosarcoma cells and tissue. More than 77% osteosarcoma specimens were stained moderately or strongly. Conclusion mAb 5D3 has high specificity for osteosarcoma. Further research on the 5D3-associated biochemical marker on osteosarcoma cells will provide possibility for early diagnosis and treatment of osteosarcoma.
Osteoarthritis (OA) is the most prevalent degenerative joint disease with multifactorial etiology caused by risk factors such as ageing, obesity and trauma. Previously, it was reported that the inhibition of microRNA-34a (miR-34a) may reduce rat chondrocyte apoptosis induced by IL-1β, whereas the molecular mechanism and the role of miR-34a in human chondrocyte as well as in OA progression remains to be determined. In the current study, using MTT, luciferase reporter assays and western blot analysis we identified that miR-34a was upregulated while silent information regulator 1 (SIRT1) was inhibited in chondrocytes from 12 OA patients compared with healthy chondrocytes from 10 trauma amputees. Overexpression of miR-34a promoted apoptosis and inhibited cell proliferation in human chondrocytes. Transfection with miR-34a mimic inhibited SIRT1 expression, which attenuated the deacetylation of p53, leading to the upregulation of Bax and downregulation of Bcl-2. Furthermore, results from the western blot analysis and luciferase reporter assay demonstrated that SIRT1 was directly regulated by miR-34a in human chondrocytes. A rat model of OA was induced through anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx). The results showed that the intra‑articular injection of lentiviral vector encoding anti-miR‑34a sequence effectively ameliorated the progression of OA. The results suggest that miR-34a has a crucial role in the pathogenesis of OA through direct regulation of the SIRT1/p53 signaling pathway and serves as a potential therapeutic target of OA.
Objective:To investigate the effective approach of isolation and cultivation of rabbit bone marrow mesenchymal stem cells(BMSCs)in Vitro and to observe their biological characteristics.Methods:Bone Marrow aspirate was harvested from femurs and tibias of the New Zealand white rabbits and BMSCs were isolated and cultured by using density gradient centrifugation and adherent culture ways.The morphological changes of the cells were observed under inverted phase contrast microscope and the growth curves of primary and passage 1,3,8 cells were drawn.The BMSCs were induced into osteogenetic and adipose cells and detected cell surface markers by flow cytometer.Results:By observing the morphology of the BMSCs,we found that the cells were fusiformate and the proliferation ability declined with time of the culture.The cells that were induced into osteogenetic cells were positive for the ALP staining and the cells were positive for the oil red O which were induced into adipose cells.What's more,CD44 expression was positive but CD34 and CD45 was negative.Conclusion:The combination of density gradient centrifugation and adhere culture is an effective method to isolate BMSCs from bone marrow aspirate and the cells were still reserved multi-directional differentiation potentiality which were cultured in Vitro.
IL-11Rα is an important cytokine receptor that links oxidative stress and compensatory proliferation. Mounting evidence has demonstrated that IL-11Rα regulates autoimmune demyelination and the invasion and proliferation of cancer cells, making it an important therapeutic target for molecular targeted therapy. Moreover, overexpression of IL-11Rα indicates a poor long-term prognosis in cancer patients. However, the expression status and its potential as a biomarker for diagnosis, tumor imaging, and prognosis in osteosarcoma remain to be determined. We report here that IL-11Rα is highly expressed in osteosarcoma and near-infrared (NIR)-labeled IL-11Rα imaging agent could detect osteosarcoma in mouse tumor xenografts. In a panel of human osteosarcoma specimens, IL-11Rα protein was positively stained in most cases by immunohistochemistry. Western blot analysis and flow cytometry showed that IL-11Rα was overexpressed in osteosarcoma SOSP-9607 cells. Cell-binding assay demonstrated specific binding of the IL-11Rα targeted imaging agent to osteosarcoma SOSP-9607 cells in vitro. In addition, administration of an IL-11Rα targeted imaging agent in a nude mice orthotopic model resulted in selective accumulation of NIR fluorescent signals in the bone tumor as well as several metabolic organs. These results indicate that IL-11Rα is a potential target for the development of molecular targeted therapy and noninvasive tumor imaging in human osteosarcoma. Furthermore, NIR-labeled IL-11Rα imaging agent is a promising lead for the development of a tumor in vivo imaging method at the molecular level in the management of human osteosarcoma.
Supplementary Data from Selective Cytotoxicity to HER2-Positive Tumor Cells by a Recombinant e23sFv-TD-tBID Protein Containing a Furin Cleavage Sequence
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