An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced

Our plain-English summary of An image cytometric technique is a concise method to detect adenoviruses and host cell proteins and to monitor the infection and cellular responses induced, by T. Morinaga, T.T.T. Nguyễn, B. Zhong, et al (Virology Journal, 2017).

The study

• Authors: T. Morinaga, T.T.T. Nguyễn, B. Zhong, M. Hanazono, M. Shingyoji, I. Sekine, Y. Tada, K. Tatsumi, H. Shimada, K. Hiroshima, M. Tagawa
• Journal: Virology Journal
• Published: 2017;14(1):219
• Read it: PubMed · Publisher · Free full text

Abstract

Background: Genetically modified adenoviruses (Ad) with preferential replications in tumor cells have been examined for a possible clinical applicability as an anti-cancer agent. A simple method to detect viral and cellular proteins is valuable to monitor the viral infections and to predict the Ad-mediated cytotoxicity.

Methods: We used type 5 Ad in which the expression of E1A gene was activated by 5'-regulatory sequences of genes that were augmented in the expression in human tumors. The Ad were further modified to have the fiber-knob region replaced with that derived from type 35 Ad. We infected human mesothelioma cells with the fiber-replaced Ad, and sequentially examined cytotoxic processes together with an expression level of the viral E1A, hexon, and cellular cleaved caspase-3 with image cytometric and Western blot analyses.

Results: The replication-competent Ad produced cytotoxicity on mesothelioma cells. The infected cells expressed E1A and hexon 24 h after the infection and then showed cleavage of caspase-3, all of which were detected with image cytometry and Western blot analysis. Image cytometry furthermore demonstrated that increased Ad doses did not enhance an expression level of E1A and hexon in an individual cell and that caspase-3-cleaved cells were found more frequently in hexon-positive cells than in E1A-positive cells. Image cytometry thus detected these molecular changes in a sensitive manner and at a single cell level. We also showed that an image cytometric technique detected expression changes of other host cell proteins, cyclin-E and phosphorylated histone H3 at a single cell level.

Conclusions: Image cytometry is a concise procedure to detect expression changes of Ad and host cell proteins at a single cell level, and is useful to analyze molecular events after the infection.

In plain English

This laboratory study in human mesothelioma cell lines tested whether image cytometry can track adenovirus (Ad) and host cell proteins in single cells. The authors used type 5 adenoviruses with the fiber-knob region of type 35 Ad, whose E1A gene was driven by survivin (AdF35/Sur) or midkine (AdF35/MK) sequences; a replication-incompetent AdF35/LacZ was used for comparison in the cell-growth test, while the image cytometry experiments compared uninfected cells with infected cells. In MSTO-211H cells, AdF35/Sur infection caused dose-dependent detachment and floating, with cell numbers falling by day 6, while AdF35/LacZ-infected cell growth was minimally suppressed. Western blotting detected E1A from 24 h, and hexon and cleaved caspase-3 from 48 h. By image cytometry E1A appeared from 12 h, hexon was detected after 24 h and peaked at 48 h, and hexon plus cleaved caspase-3 double-positive cells exceeded E1A plus cleaved caspase-3 cells. Two days after AdF35/Sur infection, cyclin E-positive MSTO-211H cells rose from 0.9 percent in uninfected cells to 14.4 percent in infected cells. The authors concluded image cytometry is a concise procedure for detecting viral and cellular proteins at a single cell level.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: In vitro laboratory experiment using human mesothelioma cell lines infected with genetically modified adenoviruses, analysed by image cytometry, immunofluorescence and Western blot
• Who was studied: Human mesothelioma cell lines NCI-H2452 and MSTO-211H, plus HEK293 packaging cells, all purchased from ATCC; no human or animal subjects
• Exposure: Replication-competent type 5 adenoviruses with type 35-derived fiber-knob, E1 driven by survivin (AdF35/Sur) or midkine (AdF35/MK) regulatory sequences, and replication-incompetent AdF35/LacZ, at 10^3 or 10^4 virus particles per cell
• Main results:
  • AdF35/Sur infection of MSTO-211H cells led to falling live cell numbers by day 6, whereas AdF35/LacZ-infected cell growth was minimally suppressed
  • AdF35/Sur-infected MSTO-211H cells detached and floated in a dose-dependent manner
  • By Western blot in MSTO-211H cells, E1A was detected from 24 h and hexon from 48 h after infection, with caspase-3 cleavage from 48 h
  • By image cytometry in MSTO-211H cells, E1A was detected as early as 12 h after infection
  • In the dual E1A and cleaved caspase-3 staining of MSTO-211H cells, double-positive cells were minimal
  • In MSTO-211H cells, hexon expression was detected after 24 h and reached its maximum at 48 h
  • Percentages of hexon plus cleaved caspase-3 double-positive MSTO-211H cells were greater than those of E1A plus cleaved caspase-3 double-positive cells
  • Western blot scarcely detected E1A in cells infected at 10^3 vp/cell, whereas image cytometry detected E1A-positive cells at the same dose
  • Hexon expression level per cell was greater in infection at 10^3 vp/cell than at 10^4 vp/cell
  • In NCI-H2452 cells infected with AdF35/Sur, E1A expression peaked on day 2 and hexon expression on day 3
  • In MSTO-211H cells infected with AdF35/MK at 10^4 vp/cell, E1A was detected at 12 h and peaked at 18 h, while cleaved caspase-3-positive cells peaked at 48 h
  • Two days after AdF35/Sur infection of MSTO-211H cells, cyclin E-positive cells rose from 0.9% in uninfected cells to 14.4% in infected cells, with phosphorylated histone H3-positive cells rising from 1.9% to 3.0% (figures given with standard errors)
• The authors' conclusion: The authors concluded that image cytometry is a simple, precise, single-cell method for detecting viral and cellular proteins, requiring few cells and useful for analysing viral infection processes and cellular responses over time
• Limitations the authors state:
  • The authors stated they could not firmly conclude a difference in sensitivity between methods because detection depends on many factors, including the affinity of the secondary antibody
  • The authors noted cytometry has limited ability to estimate numbers of infectious viral particles, since particle numbers and gene expression levels were not directly correlated

Topics

The National Library of Medicine indexes this study under subjects our site covers: Lung cancer, Mesothelioma.

Medical terms in this study, explained in our Lexicon: Mesothelioma.

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