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Alveolar Macrophages Isolated Directly From Human Cytomegalovirus (HCMV)-Seropositive Individuals Are Sites of HCMV Reactivation In Vivo.

Poole E, Juss JK, Krishna B, Herre J, Chilvers ER, Sinclair J - J. Infect. Dis. (2014)

Bottom Line: Human cytomegalovirus (HCMV) causes significant morbidity in the immunocompromised host.This has been used to support the concept that viral reactivation in HCMV carriers routinely occurs from such terminally differentiated myeloid cells in vivo.However, to date this has not been shown for in vivo-differentiated macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, United Kingdom.

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Monocytes ex vivo from a human cytomegalovirus (HCMV)-seropositive patient transcribe the HCMV latency gene UL138, and alveolar macrophages from the same donor express the HCMV lytic immediate early gene (IE) protein. Donors were tested for systemic inflammation (as revealed by the presence of C-reactive protein) and local bronchoalveolar fluid (BALF) inflammation (by means of enzyme-linked immunosorbent assay [ELISA] specific for tumor necrosis factor α [TNF-α]). A positive control for the ELISA was supernatant from monocyte-derived dendritic cells (DCs) shown in Table 2. A, Monocytes were isolated from venous blood and harvested for reverse-transcription quantitative polymerase chain reaction analysis. B, Isolated alveolar macrophages were cytospun and then fixed and stained for detection of the HCMV lytic antigen, IE. Cells were counterstained with Hoechst stain to detect nuclei.
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JIU837F3: Monocytes ex vivo from a human cytomegalovirus (HCMV)-seropositive patient transcribe the HCMV latency gene UL138, and alveolar macrophages from the same donor express the HCMV lytic immediate early gene (IE) protein. Donors were tested for systemic inflammation (as revealed by the presence of C-reactive protein) and local bronchoalveolar fluid (BALF) inflammation (by means of enzyme-linked immunosorbent assay [ELISA] specific for tumor necrosis factor α [TNF-α]). A positive control for the ELISA was supernatant from monocyte-derived dendritic cells (DCs) shown in Table 2. A, Monocytes were isolated from venous blood and harvested for reverse-transcription quantitative polymerase chain reaction analysis. B, Isolated alveolar macrophages were cytospun and then fixed and stained for detection of the HCMV lytic antigen, IE. Cells were counterstained with Hoechst stain to detect nuclei.

Mentions: We and others have previously shown that latent infection in healthy seropositive virus carriers can be assessed by the analysis of peripheral monocytes for HCMV lytic gene expression [2]. To ensure that the seropositive donor was latently infected, CD14+ monocytes were isolated from blood and analyzed for the standard hallmarks of latency: the presence of latent transcripts and the absence of lytic gene transcription. Figure 3A shows that CD14+ cells isolated from venous blood of the seropositive donor expressed the latency-associated transcript UL138 in the absence of expression of the lytic IE gene. We did not observe viral gene expression in monocytes from the seronegative donor. Alveolar macrophages from both donors were stained for detection of IE; IE expression was detectable in a few cells from the seropositive donor (Figure 3B) but in no cells from the seronegative donor (data not shown).Figure 3.


Alveolar Macrophages Isolated Directly From Human Cytomegalovirus (HCMV)-Seropositive Individuals Are Sites of HCMV Reactivation In Vivo.

Poole E, Juss JK, Krishna B, Herre J, Chilvers ER, Sinclair J - J. Infect. Dis. (2014)

Monocytes ex vivo from a human cytomegalovirus (HCMV)-seropositive patient transcribe the HCMV latency gene UL138, and alveolar macrophages from the same donor express the HCMV lytic immediate early gene (IE) protein. Donors were tested for systemic inflammation (as revealed by the presence of C-reactive protein) and local bronchoalveolar fluid (BALF) inflammation (by means of enzyme-linked immunosorbent assay [ELISA] specific for tumor necrosis factor α [TNF-α]). A positive control for the ELISA was supernatant from monocyte-derived dendritic cells (DCs) shown in Table 2. A, Monocytes were isolated from venous blood and harvested for reverse-transcription quantitative polymerase chain reaction analysis. B, Isolated alveolar macrophages were cytospun and then fixed and stained for detection of the HCMV lytic antigen, IE. Cells were counterstained with Hoechst stain to detect nuclei.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4442624&req=5

JIU837F3: Monocytes ex vivo from a human cytomegalovirus (HCMV)-seropositive patient transcribe the HCMV latency gene UL138, and alveolar macrophages from the same donor express the HCMV lytic immediate early gene (IE) protein. Donors were tested for systemic inflammation (as revealed by the presence of C-reactive protein) and local bronchoalveolar fluid (BALF) inflammation (by means of enzyme-linked immunosorbent assay [ELISA] specific for tumor necrosis factor α [TNF-α]). A positive control for the ELISA was supernatant from monocyte-derived dendritic cells (DCs) shown in Table 2. A, Monocytes were isolated from venous blood and harvested for reverse-transcription quantitative polymerase chain reaction analysis. B, Isolated alveolar macrophages were cytospun and then fixed and stained for detection of the HCMV lytic antigen, IE. Cells were counterstained with Hoechst stain to detect nuclei.
Mentions: We and others have previously shown that latent infection in healthy seropositive virus carriers can be assessed by the analysis of peripheral monocytes for HCMV lytic gene expression [2]. To ensure that the seropositive donor was latently infected, CD14+ monocytes were isolated from blood and analyzed for the standard hallmarks of latency: the presence of latent transcripts and the absence of lytic gene transcription. Figure 3A shows that CD14+ cells isolated from venous blood of the seropositive donor expressed the latency-associated transcript UL138 in the absence of expression of the lytic IE gene. We did not observe viral gene expression in monocytes from the seronegative donor. Alveolar macrophages from both donors were stained for detection of IE; IE expression was detectable in a few cells from the seropositive donor (Figure 3B) but in no cells from the seronegative donor (data not shown).Figure 3.

Bottom Line: Human cytomegalovirus (HCMV) causes significant morbidity in the immunocompromised host.This has been used to support the concept that viral reactivation in HCMV carriers routinely occurs from such terminally differentiated myeloid cells in vivo.However, to date this has not been shown for in vivo-differentiated macrophages.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, University of Cambridge, United Kingdom.

Show MeSH
Related in: MedlinePlus