Nodular B-cell aggregates associated with treatment refractory renal transplant rejection resolved by rituximab.

Acute rejection episodes leading to treatment refractory early graft loss are increasingly rare events in living related renal transplantation today. Pathophysiologic pathways often remain unsolved. We report on tubulointerstitial and vascular rejection developing within 2 weeks after transplantation in a 12-year-old boy treated with cyclosporine, mycophenolate, steroids and double blinded basiliximab. Despite steroid pulses, switch to tacrolimus and ATG serum creatinine peaked at 347 micromol/L with imminent graft loss and ongoing C4d negative cellular vascular rejection. Permanent gain of function was only achieved after a single dose of rituximab. Retrospectively CD20+ nodular B-cell aggregates could be demonstrated in all three biopsies obtained prior to rituximab and resolved concomitantly with functional improvement. Our case for the first time demonstrates resolution of nodular CD20+ infiltrates and decline of OX40, NF-kappaB and CTL transcription shortly after rituximab indicating a B-cell facilitated C4d negative pathway. Single dose rituximab may effectively reverse even long-lasting refractory rejection.

Alterations of cyclosporin A metabolism induced by mycophenolate mofetil.

Unexpectedly lower CsA 2-h levels were found in patients who received additional immunosuppression by MMF in our center. The aim of this study is to prove whether there are differences in CsA metabolization when MMF is added to treatment. In 33 children who received an immunosuppression of prednisolone and CsA as well as in 15 children who were treated with additional MMF, C2 and C0 were taken. In 11 children, full AUC of CsA were obtained. C2 levels differed significantly (p < 0.05) between patients treated with (617 +/- 230 ng/mL) and without MMF (750 +/- 271 ng/mL) but with similar CsA dosages. C2 correlated better with the AUC than CsA levels at other time points with r = 0.95. The equation AUC = 139 + 6.17 x C2 was calculated to match best in a C2-limited sampling strategy. This formula proved to be safer than equations that had been published before. According to our findings, we suspect that MMF alters the CsA metabolism. When MMF is used in pediatric transplant recipients, either target values of CsA have to be changed or patients may require higher doses of CsA.

Interleukin-2 receptor antibody-induced alterations of ciclosporin dose requirements in paediatric transplant recipients.

In a retrospective analysis of paediatric renal-transplant recipients receiving basiliximab, we noted significantly increased blood concentrations of cyclosporin, early cyclosporin toxicity, and a lower dose requirement within the first 10 days compared with controls. As the CD25 saturation fades at days 28-50, cyclosporin concentrations decline and 20% higher doses are required to maintain adequate trough concentrations. We suggest that an interleukin-2 receptor-mediated alteration of the cytochrome P450 system causes this systemic drug interaction and propose that the initial ciclosporin dose should be limited to 400 mg/m2 if used in combination with basiliximab.

Increased nuclear factor-kappaB and angiotensinogen gene expression in posttransplant recurrent focal segmental glomerulosclerosis.

In an attempt to identify potential markers of steroid-resistance in focal segmental glomerulosclerosis (FSGS) we evaluated intra-graft gene expression of IkappaBalpha, nuclear factor-kappaB (NF-kappaB), and angiotensinogen in 60 biopsies from 27 pediatric renal transplant recipients. Intra-graft NF-kappaB expression was significantly elevated in recurrent FSGS (R-FSGS) (218.3 + 55.6 ag/fg versus NON-FSGS 121.1 + 19.9, P=0.04) but not in acute rejection. NF-kappaB:IkappaBalpha ratios were higher in cadaveric donor versus living related donor recipients (15.7 + 2.8 vs. 8.8 + 1.3, respectively, P=0.015), and in African-American versus Caucasian recipients (15.6 + 2.9 vs. 9.1 + 1.3, respectively, P=0.03). Intra-graft angiotensinogen gene expression was significantly elevated in R-FSGS (30.5 + 8.8 ag/fg R-FSGS vs. 16.0 + 4.7 NON-FSGS, P=0.009). We conclude that increased NF-kappaB and angiotensinogen gene expression are associated with R-FSGS. Increased NF-kappaB:IkappaBalpha ratios are associated with cadaveric donor recipients and African-American race.

Dosing of MMF in combination with tacrolimus for steroid-resistant vascular rejection in pediatric renal allografts.

Steroid-resistant vascular rejection was treated in seven adolescent renal allograft recipients using the combination of mycophenolate mofetil (MMF) and tacrolimus (FK506). Since there are no published pediatric dose recommendations for MMF using this combination, trough concentrations and pharmacokinetic profiles were used for therapeutic drug monitoring. In order to keep the mycophenolic acid (MPA) concentrations between 2-5 micrograms/ml, mean MMF doses were reduced from 600 to 250 mg/m2 b.i.d. Apparent clearance of MPA decreased from 5 to 1 ml/min per kg within 2 weeks. Pharmacokinetic monitoring revealed substantial variability among patients of both MMF and FK506. The MPA dose ranged from 178 to 1008 mg/m2 per day to achieve an area under the curve (AUC) of 59.9 micrograms x h/ml +/- 10.5 SD (range 49-65 micrograms). FK506 dose ranged from 1.3 to 8.8 mg/m2 per day to achieve an AUC of 116 ng x h/ml +/- 27 SD (range 83-145). We recommend adjusting MMF doses using therapeutic drug monitoring.

Increased CD40 ligand gene expression during human renal and murine islet allograft rejection.

BACKGROUND: The interaction between CD40 and its ligand CD40L is essential for the development and maintenance of vigorous immunity. We have sought to determine: (i) whether a heightened level of CD40L transcripts is evident during acute allograft rejection and (ii) the kinetics of CD40L gene expression during allograft rejection. METHODS: By using quantitative reverse transcriptase-assisted polymerase chain reaction techniques, we found that heightened CD40L gene expression is a correlate of acute human renal allograft rejection (P<0.01). RESULTS: In a murine model of MHC-mismatched islet allografts, our results showed that CD40L transcripts were rarely detected at day 2 after transplantation, but were remarkably heightened at day 5 after transplantation. The transcript levels then steadily increased and peaked at the time of rejection. CONCLUSION: These data suggest that therapy aimed at blocking the CD40 to CD40L interaction should be applied during the immediate posttransplant period.

Prophylactic oral ganciclovir after renal transplantation-dosing and pharmacokinetics.

Ganciclovir alone or in combination with hyperimmunoglobulin is replacing other treatment modalities for the prophylactic treatment of cytomegalovirus (CMV) infections. No dose recommendations are available for oral ganciclovir therapy in children with impaired renal function after renal transplantation of a kidney from a CMV IgG-positive donor. We undertook a pharmacokinetic study in 14 pediatric renal transplant recipients who were CMV IgG negative and had received a graft from a CMV IgG-positive donor. We estimated the daily dosage of oral ganciclovir in relation to the glomerular filtration rate (GFR). Oral ganciclovir was administered at a starting dose of 3 x 1 g for children with a weight above 50 kg, 3 x 750 mg for children between 50 and 37.5 kg, and 3 x 500 mg for children between 37.5 and 24 kg. The starting dose was reduced by 50% for GFR values < or = 50 ml/min per 1.73 m2 and by 75% for GFR values < or = 25 ml/min per 1.73 m2. The daily dose was divided into three daily doses unless GFR was < 40 ml/ min per 1.73 m2, when only two daily doses were given. Doses were adjusted according to the measured plasma trough concentrations (c) using the simple formula: c(ganciclovir)(measured)/c(ganciclovir)(desired) = dosage rate(used)/dosage rate(adjusted). Mean stable plasma trough concentration was 0.91 +/- 0.68 microg/ml. The dosage rate, adjusted to a trough concentration of 1.0 microg/ml, correlated with the GFR. The dose per day could be calculated according to a simple equation for a GFR < 100 ml/min per 1.73 m2: dosage per day (mg/kg per day) = GFR. No CMV disease developed in any of the patients during oral ganciclovir, but 1 patient developed an acute rejection episode and a positive pp65 antigen 5 weeks after discontinuation of ganciclovir. The drug was well tolerated and without side effects.

The uretero-vesical jet as a functional diagnostic tool in childhood hydronephrosis.

Childhood hydronephrosis (HN) is accurately detected by ultrasound (US), but its functional work-up remains a domain of scintigraphy, the Whitaker-test, and clinical follow-up. We utilized color doppler US (CD-Jet) of uretero-vesical jets (UVJ) to visualize the post-obstructive ureteral flow in childhood HN. A total of 177 standardized CD-Jet were performed in 132 children aged 1 day to 14.9 years. Sixty-nine investigations in 43 patients with unilateral HN were compared with a standardized technetium 99m-mercaptotriacetylglycine nephrogram; in 10 infants both procedures were performed consecutively on the same day. UVJ were visible in 96% of all investigations. Ureteral obstruction resulted in absence of UVJ in 85% of examinations; in the remaining 15% the jet frequency was less than 10% observed in the contralateral control. Results are highly significant in both uretero-pelvic (P < 0.00007) and uretero-vesical lesions (P < 0.00005, Wilcoxon test) and are reproducible in sequential investigations. In nonobstructive distal HN the jet frequency averaged 70% of the unaffected side (P < 0.05). In proximal lesions it is equal. Jet evaluation in reflux nephropathy did not differ from controls. Compared with scintigraphy, CD-US identifies obstruction with a specificity of 94.2% and sensitivity of 94.8%. CD-US of UVJ therefore may serve as a reliable screening parameter in unilateral childhood HN.

Quantitative detection of immune activation transcripts as a diagnostic tool in kidney transplantation.

Procedures to diagnose renal allograft rejection depend upon detection of graft dysfunction and the presence of a mononuclear leukocytic infiltrate; however, the presence of a modest cellular infiltrate is often not conclusive and can be detected in non-rejecting grafts. We have pursued a molecular approach utilizing reverse transcription (RT)-PCR to test the diagnostic accuracy of multiple immune activation gene analysis as means to diagnose renal allograft rejection. The magnitude of intragraft gene expression of 15 immune activation genes was quantified by competitive RT-PCR in 60 renal allograft core biopsies obtained for surveillance or to diagnose the etiology of graft dysfunction. Results were compared with a clinicopathological analysis based upon the histological diagnosis (Banff criteria) and the response to antirejection treatment. During acute renal allograft rejection intragraft expression of the interleukin (IL)-7 (P < 0.001), IL-10 (P < 0.0001), IL-15 (P < 0.0001), Fas ligand (P < 0.0001), perforin (P < 0.0001), and granzyme B (P < 0.0015), but not IL-2, interferon gamma, or IL-4, genes is significantly heightened. Amplified RANTES and IL-8 gene transcripts are sensitive but nonspecific markers of rejection. A simultaneous RT-PCR evaluation of perforin, granzyme B, and Fas ligand identifies acute rejection, including cases with mild infiltration, with extraordinary sensitivity (100%) and specificity (100%). Effective antirejection therapy results in a rapid down-regulation of gene expression. The combined analysis of Fas ligand, perforin, and granzyme B gene expression by quantitative RT-PCR provides a reliable tool for diagnosis and follow-up of acute renal allograft rejection. Its accuracy and a potential rapid application within few hours suggest its use in the clinical management of renal transplant patients.

Intragraft IL-15 transcripts are increased in human renal allograft rejection.

IL-15, a novel growth factor made by a variety of cells, stimulates T cell proliferation in a fashion similar to IL-2. IL-2 transcripts are not routinely found in rejecting human renal allografts at the time of clinically evident rejection. However, T cell proliferation continues as the rejection progresses. We postulated that IL-15 may be actively transcribed during clinical rejection and account, at least in part, for the ongoing T cell expansion. RNA was extracted from renal biopsies and reverse transcribed to cDNA which was used as template for competitive PCR. IL-2 mRNA was detected in just 3 of the 45 biopsy samples. IL-15 transcripts were detected in all renal biopsy specimens and was significantly increased in specimens obtained from rejecting as compared with nonrejecting renal allografts. IL-15 transcription correlates with rejection and may play an important role in T cell mediated rejection.

The intragraft gene activation of markers reflecting T-cell-activation and -cytotoxicity analyzed by quantitative RT-PCR in renal transplantation.

T-cell activation is the key event in the development of acute allograft rejection and precedes clinically apparent organ damage. We have performed competitive RT-PCR to quantify the intragraft gene expression for T-cell associated cytokines (IL-2, IL-4, IL-7, IL-15), CTLA4 and cytotoxic lymphocyte specific molecules to test their potential as rejection markers and to further elucidate mechanisms involved in graft rejection. RNA was isolated from snap-frozen portions of core biopsies obtained for the evaluation of graft dysfunction in 34 adults and 8 children. Reverse transcription derived cDNA was coamplified with a known amount of a competitor (a mutated target gene fragment) and normalized for the house keeping gene GAPDH. IL-2, the principal T-cell growth factor and IL-4 were not detectable in any biopsy at the time of histologically apparent rejection. Transcripts of the novel cytokine IL-15 were found in all dysfunctioning grafts and in two donor kidneys prior to reperfusion. CTLA-4, expressed in activated T-cells after costimulation by CD28 was uniformly present post transplantation, but not in the two donor kidneys. Transcripts for IL-7 (p < 0.001), IL-15 (p < 0.0005), CTLA4 (p = 0.04), granzyme B (p < 0.00015) and perforin (p < 0.0003) showed a significant correlation to acute rejection episodes. Heightened gene expression declined rapidly after initiation of rejection treatment. Fas-ligand mRNA gene expression was upregulated in both acute and chronic rejections. While this study shows that competitive RT-PCR is a reliable diagnostic tool to detect acute rejection in renal core biopsies, a future challenge will be to identify molecular markers of evolving rejections utilizing RT-PCR in sequential samples of fine needle aspirations, urine and blood.

Increased need of erythropoietin during peritonitis in children on continuous peritoneal dialysis.

Ten continuous ambulatory peritoneal dialysis (CAPD) patients experienced 23 episodes of peritonitis and were treated with intraperitoneal (IP) antibiotics as per sensitivity report. Serum ferritin was measured before and after the treatment. In 6 patients, erythropoietin (EPO) was also measured before and after the treatment. There was a significant drop in the serum EPO levels after therapy compared to the levels before, whereas serum ferritin levels did not change.

Low-dose cyclosporin A therapy in cadaver renal transplantation in children.

Fifty-one pediatric patients undergoing a first cadaveric kidney transplantation were followed for at least 2 years after grafting. They were divided into two groups: those treated with methylprednisolone plus azathioprine (AZA) and those treated with methylprednisolone plus low-dose cyclosporin A (CyA; median dose 109 mg/m2 per day = 3.4 mg/kg per day after 1 year). The steroid dosage given was significantly lower in the second group. The 4-year graft survival rate was 68% for the AZA group and 78% for the CyA group. Renal function did not differ significantly in the two groups; after 1, 2, and 3 years, the median 24-h creatinine clearance was 79, 69, and 51 ml/min/1.73 m2, respectively, for the AZA group and 78, 63, and 68 ml/min/1.73 m2, respectively, for the CyA group. Linear growth was similar in the two groups. We conclude that in pediatric patients the results of low-dose CyA immunosuppression do not differ significantly from those obtained with AZA in terms of graft survival, renal function, or growth.