Epstein-Barr virus load for early detection of lymphoproliferative disorder in pediatric renal transplant recipients.

AIM: The aims of this study were to establish a protocol for monitoring Epstein-Barr virus (EBV) infection for identification of pediatric renal transplant recipients with a high risk of developing posttransplant lymphoproliferative disorder (PTLD) and to predict the development of PTLD. SUBJECTS AND METHODS: Peripheral blood mononuclear cells (PBMCs) and plasma EBV loads were measured by nested PCR (n-PCR) and real-time PCR (r-PCR) every 1 - 3 months after grafting in 17 pediatric recipients who were seronegative for EBV before grafting (4 with EBV-associated symptoms, including 2 with PTLD (Group A); 6 with asymptomatic persistent high EBV loads in PBMCs of > 1,000 copies/µgDNA for over 6 months (Group B); and 7 with neither EBV-associated symptoms nor persistent high EBV loads in PBMCs (Group C)). RESULTS: n-PCR revealed EBV-DNA in PBMCs from all patients. The EBV genome was present in plasma in 3 (75%), 1 (17%), and 0 (0%) in Groups A, B and C (p < 0.01 for A vs. B and A vs. C). EBV loads detected by r-PCR in PBMCs were significantly higher in Groups A (p < 0.05) and B (p < 0.01) compared to Group C. EBV genomes in plasma were detected by n- and r-PCR in only the 2 cases with PTLD. One patient with lymphadenitis in Group A and 1 patient in Group B had EBV-DNA in plasma based on n-PCR, but the viral loads using r-PCR were < 250 copies/ml. CONCLUSION: EBV loads in PBMCs alone are insufficient for predicting EBV-associated symptoms including PTLD. Plasma EBV loads (over 250 copies/ml) estimated by r-PCR may be useful to distinguish PTLD from other EBV-associated diseases or asymptomatic viremia.

Monitoring of Epstein-Barr virus load and killer T cells in pediatric renal transplant recipients.

AIM: The aim of this study is to establish a monitoring method to prevent Epstein-Barr virus (EBV)-associated symptoms including post-transplant lymphoproliferative disorder (PTLD) that occur after pediatric renal transplantation. SUBJECTS AND METHODS: Circulating EBV loads were quantified by real-time PCR every 1 - 3 months after grafting in 22 pediatric recipients (13 EBV-seronegative [R(-)] and 9 EBV-seropositive [R(+)] recipients before grafting). The peripheral blood cell populations of non-specific activated killer cells (CD8+HLA-DR+ phenotype) in 13 R(-) recipients and EBV-specific cytotoxic T cells (CTLs) reactive with a tetramer expressing HLA-A24-restricted EBV-specific antigens in 8 of 13 R(-) recipients were determined by flow cytometry. RESULTS: EBV-associated symptoms including PTLD (2 cases) were found in 4 R(-) and none of the R(+) recipients. The maximum of EBV load in the R(-) group was significantly higher that in the R(+) group. In R(-) recipients, 4 symptomatic cases had significantly more EBV genome than asymptomatic cases. EBV-specific CTLs were detected in 6 of the 8 R(-) recipients, but these CTLs could not be detected in 1 of the 2 cases at onset of PTLD. The percentage of CD8+HLA-DR+ cells was significantly higher in asymptomatic recipients than in recipients with EBV-associated symptoms whose EBV loads were over 400 copies/microg DNA. CONCLUSION: Monitoring of killer T cells and EBV loads may allow assessment of the risk of EBV-associated symptoms, and high EBV loads and low EBV-specific and/or non-specific CTL responses may be predictive for development of EBV-associated symptoms such as PTLD.

Increase of integrin-linked kinase activity in cultured podocytes upon stimulation with plasma from patients with recurrent FSGS.

Recurrent focal segmental glomerulosclerosis (FSGS) is a major challenge in the field of transplantation. Integrin-linked kinase (ILK) has emerged as a key mediator of podocyte-glomerular basement membrane (GBM) interactions. To clarify the involvement of plasma factors in FSGS recurrence, we examined the effects of plasma from FSGS patients with or without posttransplant recurrence on cultured podocytes, focusing particularly on ILK activity. Podocytes from a conditionally immortalized mouse podocyte cell line were treated with plasma from 11 FSGS patients, and ILK activity was determined using an immune complex kinase assay. Treatment with plasma from three patients with recurrence induced an increase in ILK activity. In contrast, no increase in ILK activity was observed in cultured podocytes treated with plasma from the remaining three patients with recurrence and five patients without recurrence. Cultured podocytes treated with plasma that induced ILK activity showed alterations of focal contact and detachment from the laminin matrix. In conclusion, this preliminary study provides experimental evidence suggesting the possible presence of circulating toxic factors in the plasma of some patients with recurrent FSGS, which induce an increase in podocyte ILK activity that may lead to the detachment of podocytes from the GBM.

Inherited factor H dysfunction and complement-associated glomerulonephritis in renal grafts of first and second transplantations.

A 38-yr-old man with factor H dysfunction and unknown glomerular disease received first and second renal transplantations (Tx) from living-related donors. His examination showed a low percentage activity of factor H (31%). Factor H dysfunction has been known to be associated with type II or III membranoproliferative glomerulonephritis (MPGN), haemolytic uraemic syndrome and IgA GN. The first graft from his mother showed diffuse mesangial deposit of IgA. His son has had IgA GN and his data also revealed a low percentage activity of factor H (33%). He and his son both showed a low activity of C3. Moreover, his father, who was the donor of the second Tx, had a low percentage activity of factor H (25%), and presented with mild glomerular deposit of C3 at operation, while he has been healthy through his entire 67 yr of life. Each of them had a low percentage activity of factor H. These findings through three generations suggested the inheritance of factor H dysfunction. The patient presented with proteinuria 3 months after the first Tx. At the first biopsy 30 months after the first Tx, light microscopy revealed minor glomerular abnormalities with electron dense deposits in subepithelial, intramembranous and mesangial regions, while immunofluorescence showed massive glomerular deposits of C3. In the second biopsy 51 months after the first Tx, the glomerulonephritis developed mesangial proliferation and crescent formation, accompanied by more massive C3 deposit and intramembranous, mesangial and subepithelial dense deposits. He then required redialysis. At the second and third biopsies within 2 months after the second Tx, the renal graft showed similar findings to the first biopsy after the first Tx. He perhaps presented with a recurrence of complement-associated GN, showing an atypical form of MPGN after Tx. These findings suggest that factor H dysfunction may play an important role of a certain pathogenesis of GN.

A peculiar vacuolization in the kidney transplant of a child treated with tacrolimus.

Tacrolimus (TAC) is a useful immunosuppressive agent in the prevention of rejection. However, the blood level between its therapeutic and toxic levels is narrow such that its nephrotoxicity is a problem. Moreover, its bioavailability and pharmacokinetics are highly variable. We experienced a case of acute nephrotoxicity, in which the blood level rose about 10 times above the expected level. We found a peculiar vacuolization in the transplant biopsy specimen. This change showed a marked vacuolization of the tubular cells, suggestive of acute nephrotoxicity by TAC.