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Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications
Published on: December 21, 2014
Culture of human amniotic cells: a system to study interferon production
A F Carvalho1, J R Santos, R Gentz
1Departamento de Microbiologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil.
Abstract:
This study investigated whether primary culture of human amniotic membrane cells (PCHAM) could be used as an in vitro model system for the study of interferon (IFN) production. PCHAM cells infected with Newcastle disease virus (NDV) produced the two antigenic types of IFN, previously shown in a amniotic membrane cells (HAM) system. PCHAM IFN was detected as early as 2 h after NDV infection and was composed by two antigenically distinct fractions, one neutralized with anti-HuIFN beta antibody and another that is not related to IFN beta, -alpha and -gamma. These fractions correspond respectively to 80 and 20 per cent of the IFN produced 4 h after virus induction, 55 and 45 per cent of the IFN produced from 4 to 12 h and 67 and 33 per cent of the IFN produced 12 h after virus induction. A cDNA library, established from PCHAM with or without NDV infection, was screened for IFN alpha and -beta using specific primers. The PCR product, amplified by IFN beta primers, was cloned, sequenced and expressed in Escherichia coli M15. The sequences of several cloned cDNAs were identical to HuIFN beta gene and the antiviral activity of the expressed protein was neutralized only by antiHuIFN-beta antibody. The other IFN fraction not neutralized by polyclonal antibodies anti-IFN beta, -alpha and -gamma is now being studied.
Insights
Primary culture of human amniotic membrane cells (PCHAM) effectively model interferon (IFN) production. These cells produce two distinct IFN types upon Newcastle disease virus infection, with one type identified as human interferon beta (HuIFN-beta).
Area of Science:
- Immunology
- Cell Biology
- Virology
Background:
- Interferons (IFNs) are crucial cytokines in the innate immune response to viral infections.
- Investigating novel in vitro models is essential for understanding IFN production mechanisms.
Purpose of the Study:
- To evaluate primary culture of human amniotic membrane cells (PCHAM) as an in vitro model for studying interferon production.
- To characterize the types and kinetics of IFN produced by PCHAM upon viral stimulation.
Main Methods:
- PCHAM cells were infected with Newcastle disease virus (NDV).
- Interferon production was assessed by antigenicity and neutralization assays.
- A complementary DNA (cDNA) library was constructed and screened for IFN-alpha and IFN-beta.
- IFN-beta complementary DNA (cDNA) was cloned, sequenced, and expressed in Escherichia coli.
Main Results:
- PCHAM cells produced two distinct antigenic types of IFN following NDV infection.
- IFN production was detected as early as 2 hours post-infection.
- One IFN fraction was neutralized by anti-HuIFN-beta antibodies, while the other remained uncharacterized.
- Sequencing confirmed the identity of cloned complementary DNA (cDNA) as human interferon beta (HuIFN-beta), and the expressed protein exhibited antiviral activity.
Conclusions:
- PCHAM cells represent a viable in vitro model system for studying interferon production.
- The study identified and characterized human interferon beta (HuIFN-beta) production in PCHAM cells.
- Further investigation is required to fully elucidate the nature of the second, uncharacterized IFN fraction.
