scholarly journals Production of IgY polyclonal antibody against diphtheria toxin and evaluation of its neutralization effect by Vero cell assay

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Morteza Rezaeifard ◽  
Roya Solhi ◽  
Mohammad Mohammadi ◽  
Ebrahim Abbasi ◽  
Mahdi Aminian
Keyword(s):  
Vero Cell ◽  
Diphtheria Toxin ◽  
Culture Medium ◽  
Precipitation Method ◽  
Enzyme Linked Immunosorbent Assay ◽  
Bacterial Disease ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Polyethylene Glycol Precipitation

Abstract Background Diphtheria is a bacterial disease which is caused by Corynebacterium diphtheriae. The symptoms are due to the diphtheria toxin produced by the bacteria. Antibiotic therapy and the use of diphtheria antitoxin is a recommended strategy to control diphtheria. Although mammalian antibodies are used to treat patients, IgY antibody has advantages over mammalian ones, including cost-effectiveness and production through non-invasive means. Moreover, in contrast to mammalian antibodies, IgY does not bind to the rheumatoid factor and does not activate the complement system. The objective of this study was to evaluate the in vitro neutralizing effect of IgY against diphtheria toxin. Results Anti-DT IgY was produced by immunization of the laying white leghorn chickens. Indirect enzyme-linked immunosorbent assay revealed successful immunization of the animals, and the IgY was purified with a purity of 93% via polyethylene glycol precipitation method. The neutralizing activity of the purified IgY was evaluated by Vero cell viability assay. This assay confirmed that 1.95 μg (8.6 μg/ml of culture medium) of anti-DT IgY would neutralize 10 fold of cytotoxic dose 99% of DT, which was 0.3 ng (1.33 ng/ml of culture medium). Conclusion This anti-DT IgY may be applicable for diphtheria treatment and quality controls in vaccine production.

Proliferative Effect of Tilapia Fish (Oreochromis niloticus) Lectin on BALB/c Mice Splenocytes

2019 ◽  
Vol 26 (12) ◽  
pp. 887-892
Author(s):  
Cynarha Daysy Cardoso da Silva ◽  
Cristiane Moutinho Lagos de Melo ◽  
Elba Verônica Matoso Maciel Carvalho ◽  
Mércia Andréa Lino da Silva ◽  
Rosiely Félix Bezerra ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Oreochromis Niloticus ◽  
Cytokine Production ◽  
Thymidine Incorporation ◽  
Con A ◽  
Cell Viability Assay ◽  
Proliferative Effect ◽  
Viability Assay ◽  
Apoptosis And Necrosis

Background: Lectins have been studied in recent years due to their immunomodulatory activities. Objective: We purified a lectin named OniL from tilapia fish (Oreochromis niloticus) and here we analyzed the cell proliferation and cytokine production in Balb/c mice splenocytes. Methods: Cells were stimulated in vitro in 24, 48, 72 hours and 6 days with different concentrations of OniL and Con A. Evaluation of cell proliferation was performed through [3H]-thymidine incorporation, cytokines were investigated using ELISA assay and cell viability assay was performed by investigation of damage through signals of apoptosis and necrosis. Results: OniL did not promote significant cell death, induced high mitogenic activity in relation to control and Con A and stimulated the cells to release high IL-2 and IL-6 cytokines. Conclusion: These findings suggest that, like Con A, OniL lectin can be used as a mitogenic agent in immunostimulatory assays.

scholarly journals Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy

2021 ◽  
Vol 22 (13) ◽  
pp. 7063
Author(s):  
Sharon Mordechay ◽  
Shaun Smullen ◽  
Paul Evans ◽  
Olga Genin ◽  
Mark Pines ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Motor Coordination ◽  
Mdx Mice ◽  
Cell Viability Assay ◽  
Muscle Fibrosis ◽  
Antifibrotic Therapy ◽  
Racemic Form ◽  
Viability Assay

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(−)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form’s effect. No effect was observed for (−)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (−)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (−)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

scholarly journals BDMC protects AD in vitro via AMPK and SIRT1

2020 ◽  
Vol 11 (1) ◽  
pp. 319-327
Author(s):  
Chenlin Xu ◽  
Zijian Xiao ◽  
Heng Wu ◽  
Guijuan Zhou ◽  
Duanqun He ◽  
...  
Keyword(s):  
Neurodegenerative Disorder ◽  
Neuroprotective Effect ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Activity Measurement ◽  
Therapeutic Approaches ◽  
Cell Viability Assay ◽  
Viability Assay ◽  
Compound C

AbstractBackgroundAlzheimer’s disease (AD) is a common neurodegenerative disorder without any satisfactory therapeutic approaches. AD is mainly characterized by the deposition of β-amyloid protein (Aβ) and extensive neuronal cell death. Curcumin, with anti-oxidative stress (OS) and cell apoptosis properties, plays essential roles in AD. However, whether bisdemethoxycurcumin (BDMC), a derivative of curcumin, can exert a neuroprotective effect in AD remains to be elucidated.MethodsIn this study, SK-N-SH cells were used to establish an in vitro model to investigate the effects of BDMC on the Aβ1–42-induced neurotoxicity. SK-N-SH cells were pretreated with BDMC and with or without compound C and EX527 for 30 min after co-incubation with rotenone for 24 h. Subsequently, western blotting, cell viability assay and SOD and GSH activity measurement were performed.ResultsBDMC increased the cell survival, anti-OS ability, AMPK phosphorylation levels and SIRT1 in SK-N-SH cells treated with Aβ1–42. However, after treatment with compound C, an AMPK inhibitor, and EX527, an SIRT1inhibitor, the neuroprotective roles of BDMC on SK-N-SH cells treated with Aβ1–42 were inhibited.ConclusionThese results suggest that BDMC exerts a neuroprotective role on SK-N-SH cells in vitro via AMPK/SIRT1 signaling, laying the foundation for the application of BDMC in the treatment of neurodegenerative diseases related to AMPK/SIRT1 signaling.

scholarly journals Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 508 ◽  
Author(s):  
Daniela Luz ◽  
Maria Amaral ◽  
Flavia Sacerdoti ◽  
Alan Bernal ◽  
Wagner Quintilio ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Lethal Dose ◽  
Dependent Manner ◽  
Fab Fragment ◽  
Cell Viability Assay ◽  
Cytotoxic Effects ◽  
Morphological Alterations ◽  
Viability Assay

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.

Evaluation of Paclitaxel (Taxol), Cisplatin, and the Combination Paclitaxel-Cisplatin in Ovarian Cancer in Vitro with the ATP Cell Viability Assay

1994 ◽  
Vol 53 (1) ◽  
pp. 44-49 ◽  
Author(s):  
Michael Untch ◽  
Bernd-Uwe Sevin ◽  
James P. Perras ◽  
Roberto Angioli ◽  
Andrea Untch ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Cell Viability ◽  
Cell Viability Assay ◽  
Viability Assay

scholarly journals Artemisinin Ameliorates Osteoarthritis by Inhibiting the Wnt/β-Catenin Signaling Pathway

2018 ◽  
Vol 51 (6) ◽  
pp. 2575-2590 ◽  
Author(s):  
Gang Zhong ◽  
Ruiming Liang ◽  
Jun Yao ◽  
Jia Li ◽  
Tongmeng Jiang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Cruciate Ligament ◽  
Interleukin 1 ◽  
Cartilage Degradation ◽  
Chondrocyte Apoptosis ◽  
Enzyme Linked Immunosorbent Assay ◽  
Cell Viability Assay ◽  
Necrosis Factor Alpha ◽  
Anti Inflammatory

Background/Aims: Current drug therapies for osteoarthritis (OA) are not practical because of the cytotoxicity and severe side-effects associated with most of them. Artemisinin (ART), an antimalarial agent, is well known for its safety and selectivity to kill injured cells. Based on its anti-inflammatory activity and role in the inhibition of OA-associated Wnt/β-catenin signaling pathway, which is crucial in the pathogenesis of OA, we hypothesized that ART might have an effect on OA. Methods: The chondro-protective and antiarthritic effects of ART on interleukin-1-beta (IL-1β)-induced and OA patient-derived chondrocytes were investigated in vitro using cell viability assay, glycosaminoglycan secretion, immunofluorescence, quantitative reverse transcription-polymerase chain reaction, and western blotting. We also used OA model rats constructed by anterior cruciate ligament transection and medial meniscus resection (ACLT+MMx) in the joints to investigate the effects of ART on OA by gross observation, morphological staining, immunohistochemistry, and enzyme-linked immunosorbent assay. Results: ART exhibited potent anti-inflammatory effects by inhibiting the expression of proinflammatory chemokines and cytokines, including interleukin (IL)-1β, IL-6, tumor necrosis factor alpha, and matrix metallopeptidase-13. It also showed favorable chondro-protective effect as evidenced by enhanced cell proliferation and viability, increased glycosaminoglycan deposition, prevention of chondrocyte apoptosis, and degeneration of cartilage. Further, ART inhibited OA progression and cartilage degradation via the Wnt/β-catenin signaling pathway, suggesting that it might serve as a Wnt/β-catenin antagonist to reduce inflammation and prevent cartilage degradation. Conclusion: In conclusion, ART alleviates IL-1β-mediated inflammatory response and OA progression by regulating the Wnt/β-catenin signaling pathway. Thereby, it might be developed as a potential therapeutic agent for OA.

scholarly journals SYNTHESIS AND ANTITUMOR ACTIVITY OF NEW MULTIFUNCTIONAL COUMARINS

2020 ◽  
Vol 17 (36) ◽  
pp. 871-883
Author(s):  
Moath Kahtan BASHIR ◽  
Yasser Fakri MUSTAFA ◽  
Mahmood Khudhayer OGLAH
Keyword(s):  
Schiff Base ◽  
Antitumor Activity ◽  
Human Cancer ◽  
Biological Activities ◽  
Cell Viability Assay ◽  
Chemical Structures ◽  
Viability Assay ◽  
Schiff Base Formation ◽  
Mcf 7

Cancer constitutes one of the most severe public health menaces worldwide. It is imperative to synthesize new compounds and explore their antitumor activity to find a potential resolution to this health problem. Synthesis of new scaffolds and evaluating their antitumor activity is a relevant approach for combating cancer development. Coumarins can exhibit diverse biological activities, and one of these is the antitumor activity. This study aimed to synthesize new coumarins by grafting their precursors to the aromatic amines via Schiff base formation and evaluating their introductory antitumor activity. New multifunctional coumarins (MC1-MC9) were prepared by integrating a functionalized coumarin with different toluidine derivatives via a Schiff-base linkage. Spectral characterization inspired by FTIR, 1H- and 13C- NMR spectroscopies has established the chemical structures of the synthesized products. The antitumor activity was explored in vitro versus four dominant human cancer lines, including HeLa, SKG, MCF-7, and AMN3. The outcomes acquired from the cell viability assay inspected by applying MTT dye have revealed that the synthesized multifunctional coumarins, particularly MC3, have a hopeful activity. It can be concluded that a similar trend of activity against the test cell lines was observed for the synthesized coumarins, with the best action being versus MCF-7 and the least one versus AMN3. This study not only affords a new scaffold of a significant antitumor activity but also provides some insights into its structureactivity relationship.

P13.08 Novel Thioridazine derivates: Antiproliferative and apoptosis-inducing activity on glioblastoma cells in vitro

2021 ◽  
Vol 23 (Supplement_2) ◽  
pp. ii34-ii34
Author(s):  
S G Schwab ◽  
K Sarnow ◽  
E Alme ◽  
R Goldbrunner ◽  
H Bjørsvik ◽  
...  
Keyword(s):  
Imaging System ◽  
Therapeutic Potential ◽  
Flow Cytometric Analysis ◽  
Annexin V ◽  
Therapeutic Effects ◽  
Cell Viability Assay ◽  
Selective Cytotoxicity ◽  
Viability Assay

Abstract BACKGROUND Although withdrawn from the market due to cardiotoxicity, we have shown that the antipsychotic drug Thioridazine shows chemosensitizing effects in combination with Temozolomide (TMZ) for the treatment of glioblastoma multiforme (GBM). Based on our prior observations, the aim of the presented project was through medicinal chemistry, to design and synthesize new compounds based on Thioridazines tricyclic structure, and to determine their therapeutic potential. MATERIAL AND METHODS Fourteen compounds were synthesized where variations were made within the tricyclic side chains. The newly synthesized compounds were screened for therapeutic efficacy with or without TMZ using a WST-1 cell viability assay as well as a real-time imaging system (IncuCyte). Tests were performed on both monolayer cell cultures, as well as on glioma stem cell spheroids (GSC). The therapeutic effects were also studied on human astrocytes (NHA) as well as on rat brain organoids (BO). Annexin V/propidium iodide (PI) double staining followed by flow cytometric analysis was performed after 48 hours of treatment. RESULTS Following an extensive screening, we identified two novel compounds (EA01 and EA02) that at concentrations of 4 and 9.5 µM showed a strong cytotoxicity on GBM cell lines (U-87 MG p<0,0001, U251 p<0,0001, LN18 p=0,0004) as well as on glioma stem cells (GSC) (P3 p<0,0001) compared to NHA and BOs respectively. Also, when BOs were confronted with GSC spheres in an invasion assay, a selective cytotoxicity was observed in the GSCs. Mechanistically, we show that both compounds induce apoptosis in the GBM cells. Moreover, intravenous delivery of increasing concentrations of EA01 and EA02 revealed no toxicity in animals at concentrations up to 21 mg/kg. CONCLUSION We have developed two new tricyclic therapeutic compounds that show a strong selective cytotoxicity in GBM cells with limited systemic toxicity in animals. Ongoing studies are investigating the therapeutic potential of EA01 and EA02 in orthotopic xenografts in vivo.

miR-208a-3p promotes NSCLC proliferation and migration by suppressing lncRNA-MEG3 expression

2021 ◽  
Author(s):  
Linfei Yang ◽  
Qian Li ◽  
Hai Zhong ◽  
Liang Ye ◽  
Surong Fang ◽  
...  
Keyword(s):  
Protein Extraction ◽  
Underlying Mechanism ◽  
In Vitro Assays ◽  
Cell Viability Assay ◽  
Migration Assay ◽  
Normal Tissues ◽  
Viability Assay ◽  
And Migration ◽  
Proliferation And Migration

Abstract Background The disordered expression of maternally expressed gene 3 (MEG3) has been observed in non-small-cell lung cancer (NSCLC). However, the molecular mechanism accounting for this abnormal expression is not fully understood. Methods MEG3 expression was detected by qRT-PCR in 51 cases of NSCLC and adjacent normal tissues. Then, the relationship between MEG3 and miR-208a-3p was assessed in vitro by cell viability assay, cell migration assay, protein extraction and western blot analysis. Resoults We observed that MEG3 expression was decreased in NSCLC tissues. And MEG3 expression was negatively related to lymph node metastasis and differentiation. Moreover, MEG3 expression is regulated by miR-208a-3p expression by overexpression and knockout experiments. Furthermore, we focused on the underlying mechanism of MEG3 downregulation. We found that the overexpression of miR-208a-3p reduced the level of MEG3 expression based on computational predictions and in vitro assays. Using CCK-8 and transwell migration assays, we found that the overexpression of miR-208a-3p can increased proliferation and apoptosis in NSCLC cells. Moreover, the depletion of MEG3 rescued the proliferation and migration induced by miR-208a-3p knockdown. Conclusion Taken together, the results of this study reveal that miR-208a-3p promotes NSCLC tumorigenesis by negatively regulating MEG3 expression and functions as an oncogenic miRNA in NSCLC.

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