Screening of anti-West Nile virus antibody drugs in vitro / 国际药学研究杂志

Objective: To screen anti-West Nile virus antibody drugs in vitro, using the previously established West Nile pseudovirus reporting system. Methods: At first, the enzyme-linked immunosorbent assay(ELISA)was used to detect the binding of 13 anti-West Nile virus antibody candidate strains previously obtained by the phage display technology to the target protein, West Nile virus E protein domainⅢ(DⅢ). Next, the collected cell culture supernatant containing the West Nile pseudovirus was diluted 102-fold, and the dilutions were incubated with the antibody candidates at relevant concentrations at room temperature for 1 h, respectively. Then, BHK21 or K562 cells were infected with the incubated pseudoviral supernatant+antibody mixture. After 48 hours, the infected cells were lysed to measure luciferase activity(RLU). Results: The tested 13 candidate antibodies could all recognize and bind to the West Nile virus E protein DⅢ. Among them, two antibodies(the antibodies 5 and 7)showed a good neutralizing activity and could effectively block the viral invasion upon target cells at the low concentration 5 mg/L, while two antibodies(the antibodies 3 and 8) showed an antibody-enhancing effect(ADE), which was manifested by enhancing viral infection of target cells. Conclusion: Using the West Nile pseudovirus reporting system that we established previously, we are able to obtain two anti-West Nile virus antibodies with neutralizing activity by the in vitro screening.

In vitro Mechanistic Assays of Tetracyclic Iridoid Compounds Isolated from Morinda lucida Benth in Leishmania species

Aims: This study investigates the activity of tetracyclic iridoid compounds against Leishmania spp. and the mechanism(s) of action. Study Design: An experimental study. Place and Duration: Department of Parasitology, Noguchi Memorial Institute for Medical Research, between September 2017 and July 2018. Methodology: The 50 % inhibitory concentration (IC50) of compounds against Leishmania donovani and L. major promastigotes were determined after 48 hours of incubation using the Alamar blue. Cytotoxicity of compounds was determined against cell lines using MTT assay. The anti-amastigote activity of compounds was further assessed by DAPI (4′,6-diamidino-2-phenylindole) staining. The mechanism of cell death induced by compounds was determined using nexin assay. Mitosis, cytokinesis and morphometry were monitored by DAPI and Kinetoplastid Membrane Protein (KMP) staining. Cell cycle arrest induced by compounds was analyzed by FACS. Results: Molucidin and ML-F52 inhibited the growth of promastigote in L. donovani (Molucidin; IC50 = 2.94±0.60 µM, ML-F52; IC50 = 0.91±0.50 µM) and L. major (Molucidin; IC50 = 1.85± 0.20 µM, ML-F52; IC50 = 1.77± 0.20 µM). ML-F52 had a 10-fold cytotoxic effect on parasites relative to normal cell lines. Against intracellular forms, Molucidin and ML-F52 inhibited intracellular amastigote replication and infectivity. Amphotericin B, Molucidin and ML-F52, induced a dose-dependent apoptotic effect on promastigotes. Although no change in KMP-11 expression was observed, iridoids inhibited cell division and morphological changes in promastigote cultures. Molucidin and ML-F52 induced apoptotic mechanism of cell death, inhibited cytokinesis and induced phenotypic changes in promastigotes. Molucidin further induced ‘’nectomonad-like’’ forms and loss of kDNA, ML-F52 induced ‘cell-rounding’ with loss of flagellum. Molucidin also induced cell growth arrest at G2-M phase (54.5 %). A significant induction of apoptosis (P = .05) was shown by an enhanced peak in the sub-G1 confirming the apoptotic inducing properties of iridoids. Conclusion: This study shows the anti-leishmania activity of tetracyclic iridoids which could be further investigated for the development of new chemotherapy against Leishmaniasis.

Establishment of the method to evaluate cardiac toxicity by real-time cell analysis system on human embryonic stem cells / 中国药理学通报

Aim To establish an in vitro early drug cardiac tox-icity evaluation method by human embryonic stem cells derived cardiomyocytes ( hESC-CM) and real-time cell analysis Cardio (RTCA Cardio) system. Method The hESC-CM were cultured at RTCA Cardio E-Plate 96. Impedance signals from hESC-CM were analyzed for beating rate, contraction amplitude and beating rhythm irregularity to determine the optimum inoculation density and detection duration. Based on this, we used 0. 1 % DMSO to be the solvent and quinidine (0. 2, 0. 78, 3. 13, 12. 5, 50 and 100 μmol·L - 1 ) known as affecting cardiac activity to validate this method. Result The results revealed no significant changes in the cell index (CI), transient pulse patterns, beating rate and amplitude of hESC-CM. Quinidine will affect the CI and transi-ent pulse patterns of hESC-CM and decrease the beating rate and amplitude of hESC-CM when its concentration ≥3. 13 μmol · L - 1 . And this effect is concentration-dependent, the higher the concentration,the more time they need to recover beating and the more significant the beating rate and amplitude inhibition of quinidine on hESC-CM. Conclusion The method established by hESC-CM and RTCA Cardio system can detect the effect of quinidine on the contraction of hESC-CM, and this indicates that this method has the potential to be an attractive high-throughput tool for screening potential drugs in early evaluation of drug car-diotoxicity.

Research Progress of Aplication of H295R Cell Line for Screening Environmental Steroid Hormone Disruptors / 环境与健康杂志

H295R cells can express all the key enzymes involved in the synthesis of steroid hormones,and have the ability to produce the steroid hormones found in the adult human adrenal cortex. Good correlation of response to toxicity has been reported between H295R cell line and normal adult human adrenal cells. H295R cell line can be used to evaluate effects of chemicals on gene expression,enzymatic activities and hormone production,and to explore the mode of action of chemicals on steroidgenesis. H295R cell line has been suggested as a potentially useful tool for screening environmental steroid hormone disruptors in vitro. The application of H295R cell line for screening environmental steroid hormone disruptors in vitro and its current issues are reviewed in this article.

In vitro tests for screening of immuno-modulating mycobacterial strains in leprosy.

There is an urgent need for the development of an in vitro assay for the initial screening of a large number of organisms from which potential candidates as vaccines can be identified. Our previous studies have demonstrated a crucial defect in the lepromatous macrophage. In this study by monitoring this defective macrophage response we have screened various mycobacteria for their ability to reverse the alterations induced by Mycobacterium leprae. Among the limited Mycobacteria tested Mycobacterium vaccae appears to be the most promising as an immunomodulator. Our results also indicate the need for caution in using the mouse model for this purpose.