Decoy Cells Research Articles

Characteristics of polyomavirus BK (BKPyV) infection in primary human urothelial cells

High-level polyomavirus BK (BKPyV) replication in urothelial cells is a hallmark of polyomavirus-associated hemorrhagic cystitis (PyVHC), a painful condition affecting bone marrow transplant recipients. In kidney transplant recipients, replication in tubular epithelial cells is associated with overt disease whereas high-level urothelial replication is clinically silent. We characterized BKPyV replication in primary human urothelial cells (HUCs) and compared it to replication in renal tubular epithelial cells (RPTECs). HUCs were easily infected, as shown by expression of T-antigens, VP1-3, and agnoprotein, and intranuclear virion production. Compared to RPTECs, progeny release was delayed by ≥24h and reduced. BKPyV-infected HUCs rounded up like “decoy cells” and detached without necrosis as shown by delayed cytokeratin-18 release, real-time viability monitoring and imaging. The data show that BKV infection of HUCs and RPTECs is significantly different and support the notion that PyVHC pathogenesis is not solely due to BKPyV replication, but likely requires urotoxic and immunological cofactors.

Polyomavirus (BK)-associated pleomorphic giant cell carcinoma of the urinary bladder: a case report

This report describes the morphological features of a pleomorphic giant cell carcinoma with focal trophoblastic differentiation of the urinary bladder in a male, 12 years post living related donor renal transplant. The voided urine cytology demonstrated rare decoy cells admixed with markedly atypical urothelial cell clusters, papillae and giant cells. Cystoprostatectomy demonstrated a nodular mass involving the trigone and right lateral-posterior wall, adjacent to the ureteral orifice. Hematoxylin-eosin stained sections showed two synchronous malignancies: (a) pleomorphic giant cell carcinoma with focal trophoblastic differentiation of the urinary bladder, metastatic to the omentum and (b) prostatic adenocarcinoma, Gleason score 3+4=7, involving the right prostate lobe. Strong diffuse expression of polyomavirus large T antigen was demonstrated in the primary and metastatic pleomorphic giant cell carcinoma, supporting a possible role for polyomavirus (BK) in the oncogenetic pathway. The prostatic adenocarcinoma was negative for polyomavirus large T antigen. Our findings of p63, CK7 and CK903 expression in pleomorphic giant cell carcinoma suggest that the tumor is of urothelial derivation. This is the first report describing the morphological features of urinary bladder pleomorphic giant cell carcinoma with trophoblastic differentiation, positive for polyomavirus large T antigen, arising in the background of BKV reactivation.

Experience with an alternative form of samples for external quality assessment of urinary sediment (Visual Sample EQA)

Urinary sediment components are mainly unstable, especially in their original forms. This makes the use of such material in routine external quality assessment surveys (EQAS) almost impossible. The development of an alternative EQAS for urinary sediment using photomicrographs (visual-sample EQAS) is described here, together with results, improvements and future plans. The original survey allowed a free-text interpretation, which proved to be time consuming and impractical. The next phase included a multiple choice answer (one from five alternatives). The current version challenges the participant with a list of over 50 alternatives, together with a "not-in-the-list" possibility. Certain elements are always interpreted more correctly (examples: erythrocytes, squamous cells, renal epithelial cells, bacteria--above 90%) than others (examples: leukocytes, fat droplets, certain crystals--below 55%). Pictures are included which are atypical (for example: decoy cells, which must be interpreted correctly, other artefacts). Occasionally interesting structures are included, but which are not evaluated. The two-dimensional pictures often limited the interpretation possibilities, so that in such cases more than one answer was allowed. The possibility of "online microscopy" using staple pictures is at present in evaluation. The acceptance of this type of EQAS is seen by the number of participants, at present over 500 per survey, four surveys per year.

Noninvasive tool for the diagnosis of polyomavirus BK–associated nephropathy in renal transplant recipients

Noninvasive methods can facilitate early diagnosis of BK virus (BKV) replication and guide the evaluation of BKV-associated nephropathy (BKVAN). We developed 3 noninvasive methods for BKVAN screening including quantitative polymerase chain reaction (PCR) assay for BKV DNA load in urine and plasma, and quantitative assay of urine cytology by light microscopy or electron microscopy, and used these assays concurrently with renal transplant biopsies for the evaluation of 338 patients. BKVAN was diagnosed in 24 (7.1%) of 338 renal recipients. The median level of the 3 methods was the highest in pattern B of BKVAN (P < 0.05). Using these 3 methods for pattern B of BKVAN yielded a high sensitivity of 100%. Using decoy cells without quantitation had a sensitivity of 95.8% and a specificity of 83.1% for BKVAN. The amount of decoy cells in urine samples was related to BKV DNAuria, BKV DNAemia, and the pattern of BKVAN. Using a decoy cell threshold of >5 per 10 high-power fields (HPF) had an ideal sensitivity and specificity for high-risk BKVAN and BKVAN. Using a decoy cell threshold of >20 per 10 HPF for BKVAN had a specificity of 99.7%. Quantitative assay of urine cytology is a very convenient and sensitive method for diagnosis of BKVAN, which can be deemed as an additional diagnostic method for quantitative PCR screening with increased accuracy.

Evaluation of different urine protocols and DNA extraction methods for quantitative detection of BK viruria in kidney transplant patients

Most transplant centers screen kidney transplant recipients for BK virus (BKV) infection using molecular techniques for the virus load determination. However, there is no consensus about the pre-analytical methods involved in the viral detection. In this study BK viral load was compared by the means of two urine treatment protocols (pelleted vs. whole urine) and two commercial DNA extraction kits for a quantitative PCR (qPCR) experiment. Ten patients who presented decoy cells in their urine sediment were selected for the study. Viral load was considerable higher (>1.5log) for pelleted urine, in comparison to whole urine but no significant difference was observed between the extraction kits. PCR inhibition did not occur by using pelleted urine. In order to increase test sensitivity to detect BK viruria, pelleted urine should be the preferred urine compartment for qPCR experiments.

UroVysion® multiprobe FISH in the triage of equivocal urinary cytology cases

The search for biological and clinical significance of equivocal urinary cytology has emerged as the most promising application of fluorescence in situ hybridization (FISH). Using the multiprobe UroVysion(®) assay, a negative FISH result in the presence of atypical urothelial cells favors the presence of reactive, benign alterations and helps to avoid unnecessary invasive procedures. However, a negative FISH result in case of a negative or equivocal cytology does not exclude low-grade urothelial neoplasia. Equivocal findings are a notorious problem after conservative treatment, particularly after BCG immunotherapy of carcinoma in situ, where even benign reactive changes can appear worrisome. In this situation, a positive FISH result in spite of non-high grade cytology independently indicates persistent or recurrent urothelial carcinoma. However, chromosomal abnormalities are not restricted to malignancy but may also occur in benign cells. Tetraploidy with a balanced duplication of the whole genome, or polyploidy can occur in non-neoplastic conditions of the bladder such as Decoy cells, radiotherapy-induced changes and urolithiasis. Thus, a positive FISH result in a patient with a history of pelvic irradiation does not prove cancer unless there is unequivocal 9p21 deletion. Recent studies show that an aggressive workup of patients with a suspicious cytology+positive UroVysion(®) result and negative cystoscopy is not currently justified. However, multi-target UroVysion(®) FISH remains an excellent tool to improve diagnosis in urinary cytopathology, provided that FISH results are interpreted in the light of the clinical situation, and that one reminds that FISH adds no diagnostic value in case of clearly positive, high-grade cytology.

Clinical Usefulness of BKV Viremia Monitoring by Plasma BKV Quantitative PCR

The present study investigated the clinical usefulness of plasma real-time PCR (plasma-PCR) in the prevention of BK virus-associated nephropathy (BKVAN). First, we investigated the diagnostic value of plasma, urine- PCR and urine cytology for the prediction of BKVAN retrospectively. Then we designed a prospective study of regular plasma-PCR monitoring and pre-emptive immunosuppression (IS) reduction based on the result. In the retrospective cohort, the prevalence of BKVAN was 3.7% (14/379) and the positive rate of decoy cells, urine-PCR (>1×1010 copies/mL), and plasma-PCR (>1×104 copies/mL) was 18.6%, 11.1%, and 5.5% respectively. Plasma-PCR was superior to urine-PCR or urine cytology in specificity and positive predictive value for detection of BKVAN. In the prospective study, regular monitoring of plasma-PCR detected significant BKV viremia in 8.3% (12/145) and BKVAN in 1 patient (0.6%). After IS reduction, BKV viremia was eliminated in 91.6% (11/12) within 103 days (25-254). In patients with viremia, the frequency of acute rejection did not increase and allograft function did not differ significantly compared to those in patients without viremia during the first year post-transplant (P > 0.05, in both). Plasma-PCR is a reliable method for the diagnosis of presumptive BKVAN and regular monitoring is useful to prevent the development of BKVAN.

Risk Assessment for Polyomavirus Nephropathy Using Urine Cytology and the Detection of Decoy Cells

Risk Assessment for Polyomavirus Nephropathy Using Urine Cytology and the Detection of Decoy Cells

Reply to Correspondence Regarding “Detection of Polyomavirus BK Reactivation After Renal Transplantation Using an Intensive Decoy Cell Surveillance Program is Cost Effective”

Reply to Correspondence Regarding “Detection of Polyomavirus BK Reactivation After Renal Transplantation Using an Intensive Decoy Cell Surveillance Program is Cost Effective”

Prevalence of Urinary Decoy Cells and Associated Risk Factors in a Brazilian Kidney, Pancreas, and Kidney–Pancreas Transplant Population

BackgroundPolyomavirus BK (BKV) is currently considered one of the most important infectious diseases in kidney transplants recipients. The prevalence of decoy cells (viral containing shed urothelial cells) in these patients varies between 20% and 60%. Of decoy-positive patients, 1%–8% develop BKV nephropathy, a finding that may be associated with graft failure in up to 80% of affected individuals. MethodsDecoy cells cytology is an easily performed and inexpensive assay useful for poliomavirus infection screening. Data on the prevalence of decoy cells in simultaneous pancreas–kidney or isolated pancreas recipients remains largely unreported. In the present study, we evaluated 221 patients ≥18 years old with >1 month follow-up after transplantation who had attended the outpatient clinic between September and December 2006. ResultsThe total prevalence of decoy cells was 16% (16.9% in kidney recipients, 5.9% in simultaneous kidney-pancreas recipients and 20% in pancreas alone recipients). There were no differences between patients with either positive or negative urinary cytology for decoy cells, regarding demographic (gender, age, race) or clinical (time posttransplantation, donor type [deceased vs living donation], and presence of delayed graft function or rejection, other associated viral infections and type of immunosuppressive drugs variables.

The Role of Urine Cytology for ‘Decoy Cells’ as a Screening Tool in Renal Transplant Recipients

Objective: To determine the role of urine cytology for ‘decoy cells’ as a screening tool for polyomavirus type BK (BKV) infection in renal transplant recipients. Study Design: This was a prospective cohort study of patients undergoing renal transplantation between 2006 and 2010. Results: A total of 442 patients underwent urine cytology for decoy cells, 27.8% underwent 1 examination only and 72.2% more than one. Of the 1,713 examinations reviewed, 426 (24.9%) were positive and 785 (45.8%) were negative for ‘decoy’ cells, 380 (22.2%) showed degenerated tubular cells and 122 (7.1%) were unsatisfactory for analysis. Urine cytology was found to have a specificity of 68.5%, a sensitivity of 84.6%, a positive predictive value of 21.2%, a negative predictive value of 97.8% and an overall accuracy of 69.9%. The incidence of polyomavirus nephropathy among the patients investigated was 11.8%. Of the 442 patients, 32 (7.2%) had graft loss, which was attributed to BKV nephropathy in 2 (6.2% of the 32). Conclusions: Urine cytology is an effective screening method for monitoring renal transplant patients, with high sensitivity and a high negative predictive value, and can therefore be used routinely in the follow-up of renal transplant patients.

Detection and characterization of syndecan-1-associated heparan sulfate 6-O-sulfated motifs overexpressed in multiple myeloma cells using single chain antibody variable fragments

This study was undertaken to generate human single chain variable antibody fragments (scFvs) reacting specifically against multiple myeloma (MM) cells using the phage display technique. To isolate myeloma-specific scFvs, we used a simple subtractive strategy by adsorbing the Griffin #1 antibody phage library against myeloma cells in the presence of excess decoy biotinylated HL60 cells, and then removing the unwanted decoy cells using streptavidin coated plates. From eleven scFvs that were isolated, two antibodies, D4A4 and D6B10 stained MM cell lines and patient MM cells with higher intensity than normal plasma cells. Both D4A4 and D6B10 scFvs immunoprecipitated syndecan-1 from myeloma cells and recognized sulfated motifs on syndecan-1-associated heparan sulfate (HS) chains. ScFv D4A4 competed with D6B10 for binding to MM cells. However, they differed in their fine specificities. ScFv D6B10 recognized HS 2,6-O-, N-sulfated motifs and, in contrast, binding of scFv D4A4 required N-sulfation combined with either 2-O- or 6-O-sulfation. Increased D6B10 binding on MM cells suggests that their HS chains contain a greater number of 2,6-O-, N-sulfated motifs than normal plasma cells. Since these highly sulfated motifs bind various angiogenic and growth factors and present them to their respective receptors, they could be instrumental for MM cell survival, proliferation and metastasis. Therefore, scFvs D4A4 and D6B10 provide a means to easily monitor changes in sulfation patterns of heparan sulfate during myeloma tumor progression.

Polyomavirus nephropathy – recent pathological diagnostic problems and the report from the 2011 Banff meeting

Polyomavirus nephropathy (PVN) is a cause of renal allograft dysfunction. Early detection of pathological abnormalities is not easy even in cases with urine decoy cells or polyomavirus viremia. A pathological classification for PVN was proposed by the Banff PVN working group, although the availability of the proposed schema has not been extensively evaluated in the clinical field. At the Banff meeting in 2011, the PVN working group reported the results of the evaluation on interobserver reproducibility and a clinicopathological study of PVN stages and renal outcome. Fair or good reproducibility was noted, although the PVN classification required further modification.

Detection of BK virus in urine from renal transplant subjects by mass spectrometry

BackgroundThe diagnosis and management of BK virus (BKV) reactivation following renal transplantation continues to be a significant clinical problem. Following reactivation of latent virus, impaired cellular immunity enables sustained viral replication to occur in urothelial cells, which potentially leads to the development of BKV-associated nephropathy (BKVAN). Current guidelines recommend regular surveillance for BKV reactivation through the detection of infected urothelial cells in urine (decoy cells) or viral nucleic acid in urine or blood. However, these methods have variable sensitivity and cannot routinely distinguish between different viral subtypes. We therefore asked whether mass spectrometry might be able to overcome these limitations and provide an additional non-invasive technique for the surveillance of BKV and identification of recipients at increased risk of BKVAN.ResultsHere we describe a mass spectrometry (MS)-based method for the detection of BKV derived proteins directly isolated from clinical urine samples. Peptides detected by MS derived from Viral Protein 1 (VP1) allowed differentiation between subtypes I and IV. Using this approach, we observed an association between higher decoy cell numbers and the presence of the VP1 subtype Ib-2 in urine samples derived from a cohort of 20 renal transplant recipients, consistent with the hypothesis that certain viral subtypes may be associated with more severe BKVAN.ConclusionsThis is the first study to identify BK virus proteins in clinical samples by MS and that this approach makes it possible to distinguish between different viral subtypes. Further studies are required to establish whether this information could lead to stratification of patients at risk of BKVAN, facilitate distinction between BKVAN and acute rejection (AR), and ultimately improve patient treatment and outcomes.

Clinical usefulness of BK virus plasma quantitative PCR to prevent BK virus associated nephropathy

The present study investigated the clinical usefulness of plasma real-time polymerase chain reaction (PCR) (plasma-PCR) in the prevention of BK virus-associated nephropathy (BKVAN). First, we investigated the diagnostic value of plasma BK-PCR, urine BK-PCR, and urine cytology for the prediction of BKVAN retrospectively. Then we designed a prospective study of regular plasma-PCR monitoring and pre-emptive immunosuppression (IS) reduction based on the result. In the retrospective cohort, the prevalence of BKVAN was 3.7% (14/379) and the positive rate of decoy cells, urine-PCR (>1 × 10(10) copies/ml), and plasma-PCR (>1 × 10(4) copies/ml) was 18.6%, 11.1%, and 5.5%, respectively. Plasma-PCR was superior to urine-PCR or urine cytology in specificity and positive predictive value for detection of BKVAN. In prospective study, regular monitoring of plasma-PCR detected significant BKV viremia in 8.3% (12/145) and BKVAN in 1 patient (0.6%). After IS reduction, BKV viremia was eliminated in 91.6% (11/12) within 103 days (25-254). In patients with viremia, the frequency of acute rejection did not increase and allograft function did not differ significantly compared with those in patients without viremia during the first year post-transplant (P > 0.05, in both). Plasma-PCR is useful to predict an increased risk for BKVAN, and regular monitoring is effective to prevent the development of BKVAN.

Clinical Manifestations of BK Virus Infection in Kidney Transplant Recipients: A Single Center Experience

Background: BK virus (BKV) has emerged as an important cause of graft dysfunction and failure in renal transplant recipients. Reduction of immunosuppressive therapy is accepted as the primary strategy for management of BKV infection in renal transplant recipients, a strategy which frequently results in graft dysfunction and failure. Herein, a single center experience of BKV infection in renal transplant recipients is presented with an emphasis on the management of BKV infection. Methods: We retrospectively reviewed the medical records of 107 renal transplant recipients who were treated at the outpatient clinic in Seoul National University Bundang Hospital from April 2003 to April 2011. The effects of the modification of immunosuppression on the replication of BKV and graft outcome were analyzed. Results: Of a total of 35 patient evaluated for BKV infection, 20 tested positive in at least one BK virus test. Decoy cells in urine were detected in 13 of the 20 patients. Of these patients, 7 developed BKV nephropathy. Four out of seven of the BKV nephropathy patients were diagnosed with biopsy, and the other three were diagnosed based on a high titer of BKV replication detected in plasma samples. Despite the reduction in immunosuppression and use of leflunomide in the seven BKV nephropathy patients, two patients suffered deterioration of renal function and one patient lost the graft with progressive renal dysfunction. Conclusions: BK virus nephropathy was not an uncommon disease and was a major cause of graft dysfunction or loss. Appropriate modification of immunosuppressive therapy, early in the course of BK nephropathy or before the occurrence of massive replication of BKV, is essential for the protection of renal allografts.

Cardamonin sensitizes tumour cells to TRAIL through ROS‐ and CHOP‐mediated up‐regulation of death receptors and down‐regulation of survival proteins

TNF-related apoptosis-inducing ligand (TRAIL) is currently in clinical trials as a treatment for cancer, but development of resistance is a major drawback. Thus agents that can overcome resistance to TRAIL are urgently needed. Cardamonin (2',4'-dihydroxy-6'-methoxychalcone) has been shown to affect cell growth by modulating various cell signalling pathways. Hence, we investigated the effect of cardamonin on the actions of TRAIL. The effect of cardamonin on TRAIL was measured by plasma membrane integrity, phosphatidylserine exposure, mitochondrial activity, and activation of caspase-8, caspase-9, and caspase-3 in human colon cancer cells. Cardamonin potentiated TRAIL-induced apoptosis and this correlated with up-regulation of both the TRAIL death receptor (DR) 4, 5 at mRNA and protein levels. TRAIL-decoy receptor DcR1 was down-regulated by cardamonin. Induction of DRs by cardamonin occurred in a variety of cell types. Gene silencing of the DRs by small interfering RNA (siRNA) abolished the effect of cardamonin on TRAIL-induced apoptosis, suggesting that sensitization was mediated through the DR. Induction of the DR by cardamonin was p53-independent but required CCAAT/enhancer binding protein homologous protein (CHOP); cardamonin induced CHOP, and its silencing by siRNA eliminated the induction of DR5. Cardamonin increased the production of reactive oxygen species (ROS) and quenching ROS abolished its induction of receptors and enhancement of TRAIL-induced apoptosis. Cardamonin also decreased the expression of various cell survival proteins. Cardamonin potentiates TRAIL-induced apoptosis through ROS-CHOP-mediated up-regulation of DRs, decreased expression of decoy receptor and cell survival proteins. Thus, cardamonin has the potential to make TRAIL more effective as an anticancer therapy.

Urinary decoy cell grading and its clinical implications.

BackgroundExamination of urine for decoy cells (DCs) is a useful screening test for polyomavirus (PV) activation. We explored the significance of the amount of DCs in persistent shedding, PV nephropathy and acute rejection.MethodsA case-controlled study was performed in 88 renal allograft patients who had DCs detected at least once in four or more urine samples.ResultsFifty one patients were classified into the high-grade shedding group (HG) and 37 patients into the low-grade shedding group (LG) according to DC shedding (≥10 or <10 DCs/10 high power field [HPF]). DC shedding of more than three consecutive months was significantly more prevalent in the HG as compared with their LG counterparts (p<0.0001). Urinary DCs were present for more than one year in 29.4% of the HG and 8.1% of the LG. Real-time polymerase chain reaction for PV was higher in both urine (51.4% vs. 11.1%) and plasma (9.1% vs. 0%) of the HG than the LG. The prevalence of PV nephropathy was higher in the HG than the LG (p=0.019). However, there was no significant difference in the prevalence of acute rejection.ConclusionsShedding of ≥10 DCs/10 HPF is associated with sustained shedding, polymerase chain reaction positivity and PV nephropathy, but not a predictor of acute rejection.

Decoy cells in urine cytology: A useful clue to post-transplant polyoma virus infection

Decoy cells in urine cytology: A useful clue to post-transplant polyoma virus infection

Surveillance Renal Allograft Biopsy on Diagnosis of BK Virus Nephropathy in Chinese Renal Transplant Recipients

Objective: We prospectively investigated 121 renal allograft biopsy specimens performed in our center. Methods: BKVAN was diagnosed by light microscopic examination and a positive immunohistochemistry staining of anti-SV40 large T antibody in a biopsy specimen. Results: Of the 121 patients, nine were diagnosed with BKVAN (7.4%). Nine patients whith BKVN classically presents as allograft dysfunction with an asymptomatic rise in serum creatinine, about 3 to 39 months posttransplant. Urinary decoy cells are positive in two patients (22.2%). The histologic changes of BKVN are not pathognomonic, and can be mistaken for allograft rejection, ie, tubulointerstitial nephritis with varying degrees of inflammatory infiltrates, tubulitis and tubular atrophy, and fibrosis. Typical findings on histology are focal interstitial mononuclear inflammatory cell infiltrates, presence of plasma cells, necrotic tubular epithelium, and presence of homogenous intranuclear inclusion bodies. Immunohistochemistry with SV40 staining were positive in allograft. Graft loss occurred in one patient and the other 8 showed progressive allograft dysfunction. Conclusion: The definitive diagnosis of BKV disease requires renal biopsy. Immunohistochemistry with SV40 staining has been used as an indirect method to document the presence of BKVAN.