Limits...
Tissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infection.

Borel N, Mukhopadhyay S, Kaiser C, Sullivan ED, Miller RD, Timms P, Summersgill JT, Ramirez JA, Pospischil A - BMC Infect. Dis. (2006)

Bottom Line: Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples.The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis.The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland. n.borel@access.unizh.ch

ABSTRACT

Background: Chlamydophila pneumoniae infection has been implicated as a potential risk factor for atherosclerosis, however the mechanism leading to persistent infection and its role in the disease process remains to be elucidated.

Methods: We validated the use of tissue microarray (TMA) technology, in combination with immunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raised against differentially expressed proteins under an interferon-gamma (IFN-gamma) induced model of chlamydial persistence.

Results: In the cell pellet array, we were able to identify differences in protein expression patterns between untreated and IFN-gamma treated samples. Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples. The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis. Subsequently, it was shown in a second TMA including archival atheromatous heart tissues from 12 patients undergoing heart transplantation, that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well, and were detectable morphologically within lesions by IHC.

Conclusion: TMA technology proved useful in documenting functional proteomics data with the morphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may be useful markers for the detection of persistent infection in vitro and in vivo.

Show MeSH

Related in: MedlinePlus

cell pellet array. Photomicrographs of TMA preparations of whole cell pellets showing differential expression pattern of C. pneumoniae proteins under untreated (A) and 50 U/ml IFN-γ treated (B) conditions at 48 hpi. Representative monolayers are shown to depict distinct differences in morphology and size of inclusions, as they were smaller and atypical under IFN-γ-induced persistence in comparison to the untreated monolayers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1622754&req=5

Figure 1: cell pellet array. Photomicrographs of TMA preparations of whole cell pellets showing differential expression pattern of C. pneumoniae proteins under untreated (A) and 50 U/ml IFN-γ treated (B) conditions at 48 hpi. Representative monolayers are shown to depict distinct differences in morphology and size of inclusions, as they were smaller and atypical under IFN-γ-induced persistence in comparison to the untreated monolayers.

Mentions: Results with the monoclonal antibody directed against LPS (mLPS) and the polyclonal antibody directed against chlamydial LPS and MOMP (pLPS/MOMP) were similar. Typical large, uniformly-staining chlamydial inclusions, located near the host cell nucleus, could be seen at 48 and 72 hpi in the untreated samples with the mLPS (Figure 1) and the pLPS/MOMP antibody. Results with the 5 polyclonal antisera tested, showed similar staining patterns in the untreated samples, however, not all inclusions were stained uniformly positive (Figure 1). Earlier in the chlamydial developmental cycle at 24 h, granular positive material was seen in the cytoplasm of untreated cell pellets with the mLPS antibody and the pLPS/MOMP antibody, as well as the GroEL, GroES and Pyk antibodies, and to a lesser extent, with the GspD (data not shown). All pellets at 24 hpi were negative with the Ndk antibody.


Tissue MicroArray (TMA) analysis of normal and persistent Chlamydophila pneumoniae infection.

Borel N, Mukhopadhyay S, Kaiser C, Sullivan ED, Miller RD, Timms P, Summersgill JT, Ramirez JA, Pospischil A - BMC Infect. Dis. (2006)

cell pellet array. Photomicrographs of TMA preparations of whole cell pellets showing differential expression pattern of C. pneumoniae proteins under untreated (A) and 50 U/ml IFN-γ treated (B) conditions at 48 hpi. Representative monolayers are shown to depict distinct differences in morphology and size of inclusions, as they were smaller and atypical under IFN-γ-induced persistence in comparison to the untreated monolayers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1622754&req=5

Figure 1: cell pellet array. Photomicrographs of TMA preparations of whole cell pellets showing differential expression pattern of C. pneumoniae proteins under untreated (A) and 50 U/ml IFN-γ treated (B) conditions at 48 hpi. Representative monolayers are shown to depict distinct differences in morphology and size of inclusions, as they were smaller and atypical under IFN-γ-induced persistence in comparison to the untreated monolayers.
Mentions: Results with the monoclonal antibody directed against LPS (mLPS) and the polyclonal antibody directed against chlamydial LPS and MOMP (pLPS/MOMP) were similar. Typical large, uniformly-staining chlamydial inclusions, located near the host cell nucleus, could be seen at 48 and 72 hpi in the untreated samples with the mLPS (Figure 1) and the pLPS/MOMP antibody. Results with the 5 polyclonal antisera tested, showed similar staining patterns in the untreated samples, however, not all inclusions were stained uniformly positive (Figure 1). Earlier in the chlamydial developmental cycle at 24 h, granular positive material was seen in the cytoplasm of untreated cell pellets with the mLPS antibody and the pLPS/MOMP antibody, as well as the GroEL, GroES and Pyk antibodies, and to a lesser extent, with the GspD (data not shown). All pellets at 24 hpi were negative with the Ndk antibody.

Bottom Line: Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples.The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis.The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zurich, Zurich, Switzerland. n.borel@access.unizh.ch

ABSTRACT

Background: Chlamydophila pneumoniae infection has been implicated as a potential risk factor for atherosclerosis, however the mechanism leading to persistent infection and its role in the disease process remains to be elucidated.

Methods: We validated the use of tissue microarray (TMA) technology, in combination with immunohistochemistry (IHC), to test antibodies (GroEL, GroES, GspD, Ndk and Pyk) raised against differentially expressed proteins under an interferon-gamma (IFN-gamma) induced model of chlamydial persistence.

Results: In the cell pellet array, we were able to identify differences in protein expression patterns between untreated and IFN-gamma treated samples. Typical, large chlamydial inclusions could be observed in the untreated samples with all antibodies, whereas the number of inclusions were decreased and were smaller and atypical in shape in the IFN-gamma treated samples. The staining results obtained with the TMA method were generally similar to the changes observed between normal and IFN-gamma persistence using proteomic analysis. Subsequently, it was shown in a second TMA including archival atheromatous heart tissues from 12 patients undergoing heart transplantation, that GroEL, GroES, GspD and Pyk were expressed in atheromatous heart tissue specimens as well, and were detectable morphologically within lesions by IHC.

Conclusion: TMA technology proved useful in documenting functional proteomics data with the morphologic distribution of GroEL, GroES, GspD, Ndk and Pyk within formalin-fixed, paraffin-embedded cell pellets and tissues from patients with severe coronary atherosclerosis. The antibodies GroEL and GroES, which were upregulated under persistence in proteomic analysis, displayed positive reaction in atheromatous heart tissue from 10 out of 12 patients. These may be useful markers for the detection of persistent infection in vitro and in vivo.

Show MeSH
Related in: MedlinePlus