Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells
Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV an...
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MDPI AG
2023-07-01
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| Online Access: | https://www.mdpi.com/2075-1729/13/7/1526 |
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| author | Sahra Pajenda Zsofia Hevesi Michael Eder Daniela Gerges Monika Aiad Oliver Koldyka Wolfgang Winnicki Ludwig Wagner Farsad Eskandary Alice Schmidt |
| author_facet | Sahra Pajenda Zsofia Hevesi Michael Eder Daniela Gerges Monika Aiad Oliver Koldyka Wolfgang Winnicki Ludwig Wagner Farsad Eskandary Alice Schmidt |
| author_sort | Sahra Pajenda |
| collection | DOAJ |
| description | Decoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6–32% of VP1<sup>+</sup> cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68–94% of them losing parts of their DNA and exhibiting membrane damage, appearing as “ghost cells” but still VP1<sup>+</sup>. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression. |
| first_indexed | 2024-03-11T00:53:27Z |
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| institution | Directory Open Access Journal |
| issn | 2075-1729 |
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| spelling | doaj.art-0b298165ae3944ab985a04d3dae53fd22023-11-18T20:09:43ZengMDPI AGLife2075-17292023-07-01137152610.3390/life13071526Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy CellsSahra Pajenda0Zsofia Hevesi1Michael Eder2Daniela Gerges3Monika Aiad4Oliver Koldyka5Wolfgang Winnicki6Ludwig Wagner7Farsad Eskandary8Alice Schmidt9Division of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaCenter for Brain Research, Medical University of Vienna, 1090 Vienna, AustriaDivision of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDivision of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDivision of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDivision of Endocrinology and Metabolism, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDivision of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDivision of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDivision of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDivision of Nephrology and Dialysis, Department of Internal Medicine III, Medical University of Vienna, 1090 Vienna, AustriaDecoy cells that can be detected in the urine sediment of immunosuppressed patients are often caused by the uncontrolled replication of polyomaviruses, such as BK-Virus (BKV) and John Cunningham (JC)-Virus (JCV), within the upper urinary tract. Due to the wide availability of highly sensitive BKV and JCV PCR, the diagnostic utility of screening for decoy cells in urine as an indicator of polyomavirus-associated nephropathy (PyVAN) has been questioned by some institutions. We hypothesize that specific staining of different infection time-dependent BKV-specific antigens in urine sediment could allow cell-specific mapping of antigen expression during decoy cell development. Urine sediment cells from six kidney transplant recipients (five males, one female) were stained for the presence of the early BKV gene transcript lTag and the major viral capsid protein VP1 using monospecific antibodies, monoclonal antibodies and confocal microscopy. For this purpose, cyto-preparations were prepared and the BK polyoma genotype was determined by sequencing the PCR-amplified coding region of the VP1 protein. lTag staining began at specific sites in the nucleus and spread across the nucleus in a cobweb-like pattern as the size of the nucleus increased. It spread into the cytosol as soon as the nuclear membrane was fragmented or dissolved, as in apoptosis or in the metaphase of the cell cycle. In comparison, we observed that VP1 staining started in the nuclear region and accumulated at the nuclear edge in 6–32% of VP1<sup>+</sup> cells. The staining traveled through the cytosol of the proximal tubule cell and reached high intensities at the cytosol before spreading to the surrounding area in the form of exosome-like particles. The spreading virus-containing particles adhered to surrounding cells, including erythrocytes. VP1-positive proximal tubule cells contain apoptotic bodies, with 68–94% of them losing parts of their DNA and exhibiting membrane damage, appearing as “ghost cells” but still VP1<sup>+</sup>. Specific polyoma staining of urine sediment cells can help determine and enumerate exfoliation of BKV-positive cells based on VP1 staining, which exceeds single-face decoy staining in terms of accuracy. Furthermore, our staining approaches might serve as an early readout in primary diagnostics and for the evaluation of treatment responses in the setting of reduced immunosuppression.https://www.mdpi.com/2075-1729/13/7/1526BK-polyomavirusdecoy cellpolyoma nephropathylarge T antigenVP1 |
| spellingShingle | Sahra Pajenda Zsofia Hevesi Michael Eder Daniela Gerges Monika Aiad Oliver Koldyka Wolfgang Winnicki Ludwig Wagner Farsad Eskandary Alice Schmidt Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells Life BK-polyomavirus decoy cell polyoma nephropathy large T antigen VP1 |
| title | Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells |
| title_full | Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells |
| title_fullStr | Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells |
| title_full_unstemmed | Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells |
| title_short | Lessons from Polyomavirus Immunofluorescence Staining of Urinary Decoy Cells |
| title_sort | lessons from polyomavirus immunofluorescence staining of urinary decoy cells |
| topic | BK-polyomavirus decoy cell polyoma nephropathy large T antigen VP1 |
| url | https://www.mdpi.com/2075-1729/13/7/1526 |
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