Interferon Inhibition Enhances the Pilot-Scale Production of Rabies Virus in Human Diploid MRC-5 Cells

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-β response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5IFNAR1−), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5IFNAR1− cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production.

Tingkat Pengetahuan Mahasiswa Fakultas Kedokteran Universitas Yarsi Mengenai Penggunaan Human Diploid Cell Dalam Proses Produksi Vaksin Polio

Latar Belakang : Vaksin merupakan suspensi mikroorganisme yang dilemahkan atau dimatikan, atau protein antikgenik dari berbagai organisme tadi yang diberikan untuk mencegah, meringankan, atau mengobati penyakit-penyakit menular. Vaksin pertama kali tercatat pada tahun 1769, yang dipublikasikan oleh Edward Jenner, yaitu specimen yang berasal dari lesi lengan seseorang yang terinfeksi Cowpox. Human Diploid Cells (HDC) merupakan salah satu sel yang digunakan untuk mengkultur virus yang akan dijadikan vaksin. HDC yang berasal dari aborsi manusia ini banyak digunakan untuk mengkultur virus Polio IPV dan OPV, Rabies, Rubella, Measles, Varicella-Zooster, dan Hepatitis A. Tujuan : Vaksin polio merupakan vaksin yang diwajibkan pada anak yang dijadwalkan dari Ikatan Dokter Anak Indonesia (IDAI) yang dibagi menjadi dua jenis, IPV (Inactivated Polio Vaccine) dan OPV (Oral Polio Vaccine). Metode : Jenis Penelitian yang digunakan adalah deskriptif dengan pendekatan cross sectional menggunakan kuesioner. Populasi yang digunakan adalah mahasisa Fakultas Kedokteran Universitas YARSI tahun pertama dan tahun ketiga yang memenuhi syarat. Cara pemilihan sampel dengan simple random sampling. Hasil : Penelitian yang dilaksanakan selama 3 hari dengan menggunakan kuesioner, dari 100 responden didapatkan persentase jumlah kuesioner Pengetahuan mengenai Human Diploid Cell berdasarkan Tingkat Pendidikan didapatkan pengetahuan baik sebanyak 5% pada tahun ketiga dan 7% pada tahun pertama. Pengetahuan cukup sebanyak 23% pada tingkat ketiga dan 28% pada tahun pertama. Pengetahuan kurang sebanyak 9% pada tingkat ketiga dan 28% pada tahun pertama. Persentase jumlah kuesioner Pengetahuan mengenai Polio berdasarkan Tingkat Pendidikan didapatkan pengetahuan baik sebanyak 15% pada tahun ketiga dan 19% pada tahun pertama. Pengetahuan cukup sebanyak 18% pada tingkat ketiga dan 31% pada tahun pertama. Pengetahuan kurang sebanyak 4% pada tingkat ketiga dan 13% pada tahun pertama. Kesimpulan : Tidak terdapat hubungan antara tingkat pendidikan dengan pengetahuan mengenai Human Diploid Cell dalam vaksin Polio. Dalam pandangan Islam, penggunaan vaksin Polio hukumnya mubah karena prinsip Dharuriyat bertujuan untuk mempertahankan nyawa atau Hifdz an-nafs anak dari ancaman penyakit.

TRANSDISCIPLINARY APPROACH IN FREE RADICAL THEORY OF AGING. CONTRIBUTION OF NIKOLAY M. EMANUEL AND HIS SCIENTIFIC SCHOOL TO GERONTOLOGY

Работа представляет собой обзор научных исследований влияния антиоксидантов-геропротекторов на старение экспериментальных животных и репликативное старение диплоидных клеток человека, выполненных в отделе кинетики химических и биологических процессов «ХИМБИО» Института химической физики АН СССР под руководством академика Николая Марковича Эмануэля в 1960- 1980- е гг. В работах Н.М. Эмануэля и сотрудников было установлено неизвестное ранее явление взаимодействия ингибиторов свободнорадикальных реакций в процессах окисления органических веществ, заключающееся в регенерации более эффективного ингибитора вследствие переноса атома водорода к его радикалу от молекулы менее эффективного ингибитора. Антиоксиданты поливалентны и могут влиять одновременно на многие процессы старения. Данные научной школы Н.М. Эмануэля по увеличению средней продолжительности жизни на 25,3 % и максимальной продолжительности мышей на 55,8 % под действием антиоксидантов, полученные в результате хорошо обоснованных экспериментальных и теоретических исследований, явились весомым аргументом в пользу свободнорадикальной теории старения. The work is aimed to review the results of scientific studies of the effect of antioxidants-geroprotectors on the aging of experimental animals and the replicative aging of human diploid cells, carried out in the Department of Kinetics of Chemical and Biological Processes «KHIMBIO» of the Institute of Chemical Physics of the USSR Academy of Sciences under the leadership of academician Nikolay Markovich Emanuel in the 1960-1980s after pioneer work by D. Harman. By N.M. Emanuel and colleagues, it was established a previously unknown phenomenon of radical interaction of inhibitors in the oxidation of organic substances, which consists in the regeneration of a more effective inhibitor due to the transfer of a hydrogen atom to its free radical from a molecule of a less effective inhibitor. Antioxidants are polyvalent and can simultaneously affect many stages of aging processes. Data from the N.M. Emanuel scientific school on the increase of the average lifespan of mice by 25,3 % and their maximum lifespan by 55,8 % using antioxidants, discovered at the Institute of Chemical Physics of the USSR Academy of Sciences as a result of well-founded experimental and theoretical studies, became a powerful argument in favor of the free radical theory of aging in 1970-ties. This was further promoted by approaches based on the theory of reliability, the damage theory, and as well as an approach based on oxidative activation of the Nrf2 signaling pathway, which maintains the «nucleophilic tone» of protective oxidoreductases.

Imputation-free reconstructions of three-dimensional chromosome architectures in human diploid single-cells using allele-specified contacts

The sparseness of chromosomal contact information and the presence of homologous chromosomes with very similar nucleotide sequences make Hi-C analysis difficult. We propose a new algorithm using allele-specific single-nucleotide variations (SNVs) to reconstruct the three-dimensional (3D) chromosomal architectures from the Hi-C dataset of single diploid cells. Our algorithm has a function to discriminate SNVs specifically found between homologous chromosomes to our “recurrence plot”-based algorithm to estimate the 3D chromosome structure, which does not require imputation for ambiguous segment information. The new algorithm can efficiently reconstruct 3D chromosomal structures in single human diploid cells by employing only Hi-C segment pairs containing allele-specific SNVs. The datasets of the remaining pairs of segments without allele-specific SNVs are used to validate the estimated chromosome structure. This approach was used to reconstruct the 3D structures of human chromosomes in single diploid cells at a 1-Mb resolution. Introducing a subsequent mathematical measure further improved the resolution to 40-kb or 100-kb. The reconstruction data reveals that human chromosomes form chromosomal territories and take fractal structures where the mean dimension is a non-integer value. We also validate our approach by estimating 3D protein/polymer structures.

Quantitative analysis of CRISPR-del for complete gene knockout in human diploid cells

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion/deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked-out by the indel- based gene disruption. In this study, we optimized CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform complete gene knockout in non-transformed human diploid RPE1 cells. By improving several procedures, the optimized CRISPR-del allowed us to generate knockout cell lines harboring bi-allelic large chromosomal deletions in a high-throughput manner. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the ability of this method to introduce a large chromosomal deletion enables the generation of a model cell line having a bi- allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the optimized CRISPR-del is a high-throughput method for performing complete gene knockout in RPE1 cells.

Intrinsic Oncolytic Activity of Hoshino Mumps Virus Vaccine Strain Against Human Fibrosarcoma and Cervical Cancer Cell Lines

Background: The use of oncolytic viruses as therapeutic agents is a promising treatment for various human cancers. Several viruses have been extensively examined to achieve tumor cell death. Objectives: This study aimed at evaluating the natural oncolytic activity of mumps Hoshino vaccine strain against two human cancer cell lines, that is, HT1080 fibrosarcoma and HeLa cervical adenocarcinoma cell lines. Methods: The cytolytic activity of the virus was evaluated using an MTT assay. Apoptosis was detected by Annexin-V/propidium iodide (PI) staining and analyzed via flow cytometry. To indicate viral replication in vivo, nude mice with HeLa heterografts were treated with the Hoshino strain of mumps virus. Results: It was found that human fibrosarcoma and cervical cells were more sensitive to the mumps Hoshino strain, even at a very low multiplicity of infection (MOI) compared to normal human diploid cells. The results also showed that the Hoshino strain induced apoptosis in both cancer cells. A preliminary in vivo study revealed the significant suppression of tumor growth in the group treated with the mumps Hoshino strain compared to the control group. Conclusions: The Hoshino vaccine strain of mumps virus showed promising oncolytic activities against human fibrosarcoma and cervical adenocarcinoma cells.

Antimicrobial Wound Dressings as Potential Materials for Skin Tissue Regeneration

The most important properties of performant wound dressings are biocompatibility, the ability to retain large amount of exudate and to avoid complications related with persistent infection which could lead to delayed wound healing. This research aimed to obtain and characterize a new type of antimicrobial dressings, based on zinc oxide/sodium alginate/polyvinyl alcohol (PVA). Zinc oxide nanostructures, obtained with different morphology and grain size by hydrothermal and polyol methods, are used as antimicrobial agents along with sodium alginate, which is used to improve the biocompatibility of the dressing. The nanofiber dressing was obtained through the electrospinning method. Characterization techniques such as X-ray diffraction (XRD) and scanning electron microscopy (SEM) were performed to determine the structural and morphological properties of the obtained powders and composite fibers. Their antimicrobial activity was tested against Gram negative Escherichia coli (E. coli), Gram positive Staphylococcus aureus (S. aureus) bacteria and Candida albicans (C. albicans) yeast strains. The in vitro biocompatibility of the obtained composites was tested on human diploid cells. The obtained results suggest that the composite fibers based on zinc oxide and alginate are suitable for antimicrobial protection, are not toxic and may be useful for skin tissue regeneration if applied as a dressing.