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.

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.

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.

Effect of pre-and postoperative plasmapheresis on posttransplant recurrence of focal segmental glomerulosclerosis in children.

BACKGROUND: Posttransplant recurrence is frequent in patients who received renal transplantation for focal segmental glomerulosclerosis (FSGS). The recurrence has been ascribed to a circulating permeability factor or factors. We have used plasmapheresis (PP) to treat recurrent FSGS and also studied whether preoperative PP is effective in preventing recurrence of FSGS. METHODS: We retrospectively analyzed 21 allografts of 20 patients with nephrotic syndrome and biopsyproven FSGS. They were divided into two groups depending on whether they had prophylactic PP; a prophylactic (n=15) and a nonprophylactic group (n=6). PP was performed two to three times prophylactically and therapeutically until proteinuria was markedly reduced. In each session, 50-75 ml/kg of the patient's plasma was exchanged with 5-8% albumin. RESULTS: FSGS recurred in 9 of 21 allografts, 4 of 6 in the nonprophylactic group, and 5 of 15 in the prophylactic group. Therapeutic PP was performed in seven of nine recurrent patients without definite adverse effect, with satisfactory results except in one patient. Children lost proteinuria after 6 to > 100 sessions of PP and the number correlated with the pretreatment level of proteinuria. The mean follow-up periods were 62.7 and 41.6 months for the prophylactic and nonprophylactic groups, respectively. At the last follow-up, 66.7% of relapsing and 81.8% of nonrelapsing patients had a functioning graft. CONCLUSION: PP appears to be effective for the prevention and treatment of posttransplant recurrence of FSGS, although further consideration of cost/benefit and risks is required before a conclusive judgement can be made.

[Structure of segmental tuft lesions in childhood IgA nephropathy--three dimensional analysis indicates development of segmental lesions].

The mechanisms responsible for the formation and development of segmental lesions of IgA nephropathy in children were studied by analysis of three dimensional reconstruction. Forty-eight segmental lesions from 15 cases (diffuse proliferative glomerulonephritis (DPGN) in 13 cases, focal glomerulonephritis (FGN) in 2 cases) were examined by light microscopy by analysis of serial sections (in average 26 sections/glomerulus). In tuft, three types of tuft lesions were defined by their chronisity: 1) endocapillary proliferation including exudative changes, 2) mesangial hypercellularity, 3) deposition of mesangial matrix with sclerosis. Extra-capillary lesions, namely crescent were also defined by their chronisity: 1) cellular, 2) fibrocellular, 3) fibrous. The structural relation in each tuft and endocapillary lesions were observed. Endocapillary proliferative lesions of tufts were closely associated with cellular crescent of extra-capillary lesions. In fibrocellular and fibrous crescents, the frequency of endocapillary proliferation in tufts were reduced, while the association of mesangial proliferation and sclerosis were increased. Nevertheless, endocapillary proliferation of tufts were still observed in 33% of fibrous crescent. We concluded that segmental lesions were originated by endocapillary tuft lesions leading to cellular crescents. The multifocal and repeated attack of endocapillary proliferative lesions within segmental sclerosis promotes further development of glomerular sclerosis in IgA nephropathy in children.

[Congenital multiple anomaly syndrome with recurrent vomiting accompanied with latent SIADH; a case report].

A 13-year-old girl with multiple minor anomalies and severe mental retardation had recurrent episodes of severe vomiting. At each episode, marked elevations of plasma ADH, ACTH, cortisol and salivary type amylase were found with reduction of serum Na level and osmolarity. This case is similar to that with periodic ACTH-ADH discharge syndrome (Sato). However, she had underlying disease, and neither hypertension nor depressive state was observed. Latent SIADH was detected by water loading test. After DZP administration, ADH secretion was suppressed in this test, and actually the duration of each attack was shortened. We considered that her vomiting was closely related with hypothalamic dysfunction, especially latent SIADH.

[Clinical study of 18 pediatric cadaveric renal transplantations: organ sharing in pediatric renal transplantation after enforcement of the organ transplant law in Japan].

Renal transplantation is considered to be the optimal replacement therapy for children with end-stage renal disease. However, the number of pediatric renal transplants in Japan is much lower than in the USA and/or Europe. Since October 1997, pediatric(< 15 years) recipients are given priority over adult recipients for organ sharing, only if one or two HLA-DR antigen(s) are matched between the recipient and pediatric(< 15 years) donor. However, the number of pediatric transplants is not increasing. One hundred and twenty-four pediatric renal transplantations were performed in Tokyo Women's Medical University between 1983 and 1999, of which 18(14.5%) were cadaveric transplants and the others (106, 85.5%) were living-related transplants. We examined 18 pediatric cadaveric renal transplantations. Seven patients received their graft from pediatric donors less than 15 years of age and 11 from adult donors. The mean age at transplantation was 13.2 years (range 4.5-18.7 years). Major etiologies of renal disease are hereditary renal disease(38.8%), chronic glomerulonephritis(33.3%), and focal segmental glomerulosclerosis[FSGS] (16.7%). Zero matches in HLA-DR locus were observed in 72.2%. Patient survival rate was 100%. Graft survival rates at 1 and 5 years after transplantation were 83% and 64% successively. There was no significant difference between the graft survival of cadaveric and living-related transplantation at 1 and 5 years. All 5 patients who received their graft between 1994 and 1998 have maintained normal graft function. Causes of their graft loss were chronic rejection in 3, recurrence of FSGS in 2, primary non-function in 1, and graft thrombosis in 1. Donor age and HLA-DR mismatching did not affect the outcome. We propose that pediatric renal grafts should be provided to children with priority, regardless of their HLA-A, B and HLA-DR matching.

Macrophages in childhood IgA nephropathy.

The role of glomerular macrophages in IgA nephropathy in children was investigated using a new monoclonal antibody (KP1) as a probe. The average number of glomerular macrophages per patient (ANM/P) was closely correlated with the degree of hematuria (P < 0.01) as well as with the degree of leukocyturia (P < 0.01) in the absence of any correlation with proteinuria, serum IgA levels or the interval between the detection of urine abnormalities and renal biopsy. ANM/P was significantly higher in patients diagnosed pathologically as having focal and diffuse proliferative glomerulonephritis than in patients with minor glomerular abnormalities or advanced sclerosis (P < 0.05). Among various types of glomerular morphology in individual patients, macrophages predominantly infiltrated glomeruli with cell-proliferative lesions despite an absence of any increase in glomeruli with minor abnormalities or with sclerosis. Macrophages were mainly localized within the capillary lumen in association with endocapillary proliferative lesions (tuft necrosis), they accumulated in areas of mesangial proliferation, and they were attached to Bowman's capsule in segmental lesions. Macrophages were less evident in sclerosis. Furthermore, ultrastructural analysis revealed macrophages in the paramesangial areas in close proximity to lytic changes in the glomerular basement membrane and effacement of epithelial foot processes. In addition, some cases in repeat biopsy shows prolonged or increased values of ANM/P after several years of interval in association with progression of proliferative lesions. These results suggest that macrophages infiltrate glomeruli during acute glomerular inflammation, and that they are involved in mesangial proliferation or the development of extracapillary lesions in the absence of apparent clinical symptoms. Furthermore, recurrence or prolonged infiltration may promote progression of IgA nephropathy.

Antineutrophil cytoplasmic antibody-positive crescentic glomerulonephritis associated with propylthiouracil therapy.

A teenage girl with crescentic glomerulonephritis had antineutrophil cytoplasmic antibody (ANCA) detected after she had received propylthiouracil (PTU) for hyperthyroidism without cutaneous vasculitis. ANCA was detected on admission; renal biopsy showed crescentic glomerulonephritis with focal necrotizing glomerulonephritis but no immune deposits. Administration of steroid and decreasing the dose of PTU produced a good clinical response and the ANCA disappeared. It was concluded that ANCA is closely related to the pathogenesis of crescentic glomerulonephritis and that treatment with PTU appeared to induce ANCA.

Cryosupernatant plasma exchange in the treatment of antiphospholipid antibody syndrome with lupus nephritis.

We report a case of a 22-year-old female with antiphospholipid antibody syndrome (APS) associated with systemic lupus erythematosus in whom cryosupernatant plasma exchange was effective and improved both the refractory venous thrombosis in her legs and relapsing thrombocytopenia. A renal biopsy specimen showed not only features of active lupus nephritis but also renal arteriolar thrombosis which is considered to be a type of thrombotic microangiopathy (TMA). Because a pathological role of unusually large von Willebrand factor (vWF) multimers has been reported in patients with TMA including thrombotic thrombocytopenic purpura, plasma exchange using replacement with cryosupernatant, which is free of unusually large vWF multimers, is likely to be an option of treatment modality for patients with refractory and chronic relapsing APS manifesting TMA.

ABO-incompatible pediatric kidney transplantation in a single-center trial.

We have performed ten pediatric kidney transplantations from living-related ABO-incompatible donors. All patients underwent preoperative plasmapheresis with or without immunoadsorption to reduce isoagglutinin. Primary immunosuppression consisted of methyl-prednisolone, cyclosporin or tacrolimus, azathioprine, anti-lymphocyte globulin, and/or deoxyspergualin. At transplantation splenectomy was simultaneously performed in all patients. Median follow-up is 65 months (range 4-95 months). The patient and graft survival rates are 100% to date. Post-transplantation isoagglutinin titers did not increase more than 1:32, except for 1 patient, without uncontrollable vascular rejection episodes. Despite the heavy immunosuppressive regimen, cytomegalovirus infection occurred in only three patients, who were successfully treated with ganciclovir and cytomegalovirus high-titer gamma globulin. Our small series clearly shows that the preoperative reduction of isoagglutinin, splenectomy, and strict immunosuppressive therapy lead to successful long-term results in children.