Luca Dello Strologo

Comparison between SLC3A1 and SLC7A9 Cystinuria Patients and Carriers: A Need for a New Classification

Recent developments in the genetics and physiology of cystinuria do not support the traditional classification, which is based on the excretion of cystine and dibasic amino acids in obligate heterozygotes. Mutations of only two genes (SLC3A1 and SLC7A9), identified by the International Cystinuria Consortium (ICC), have been found to be responsible for all three types of the disease. The ICC set up a multinational database and collected genetic and clinical data from 224 patients affected by cystinuria, 125 with full genotype definition. Amino acid urinary excretion patterns of 189 heterozygotes with genetic definition and of 83 healthy controls were also included. All SLC3A1 carriers and 14% of SLC7A9 carriers showed a normal amino acid urinary pattern (i.e., type I phenotype). The rest of the SLC7A9 carriers showed phenotype non-I (type III, 80.5%; type II, 5.5%). This makes the traditional classification imprecise. A new classification is needed: type A, due to two mutations of SLC3A1 (rBAT) on chromosome 2 (45.2% in our database); type B, due to two mutations of SLC7A9 on chromosome 19 (53.2% in this series); and a possible third type, AB (1.6%), with one mutation on each of the above-mentioned genes. Clinical data show that cystinuria is more severe in males than in females. The two types of cystinuria (A and B) had a similar outcome in this retrospective study, but the effect of the treatment could not be analyzed. Stone events do not correlate with amino acid urinary excretion. Renal function was clearly impaired in 17% of the patients.

Feeding dysfunction in infants with severe chronic renal failure after long-term nasogastric tubefeeding

Abstract.Nasogastric tube feeding (NGTF) is frequently necessary to overcome the inadequate caloric intake of children with severe chronic renal failure (CRF). In a multicenter retrospective study, we evaluated feeding dysfunction after tube feeding withdrawal in children with severe CRF who started long-term enteral nutrition early in childhood. We considered, almost exclusively, infants who had started NGTF very early and continued to be tube fed for at least 9 months. Twelve patients were included in the study: 8 showed significant and persistent eating difficulties, with difficulties in chewing and swallowing in 7 and food refusal in 6. For 2 patients “panic attacks” from swallowing were repeatedly reported. These problems persisted for more than year in 5 patients and between 1 and 6 months in 4. The possible feeding difficulties that may follow NTGF must be carefully evaluated. A possible means of overcoming these difficulties might include: encouraging the use of a pacifier, proposing water for spontaneous assumption, leaving the child the possibility of eating food spontaneously during the daytime, and increased support for the parents during weaning. These need prospective study.

Inhibition of B-Cell Proliferation and Antibody Production by Mesenchymal Stromal Cells Is Mediated by T Cells

Bone marrow (BM)-derived mesenchymal stromal cells (MSCs), endowed with immunosuppressive and anti-inflammatory properties, represent a promising tool in immunoregulatory and regenerative cell therapy. Clarifying the interactions between MSCs and B-lymphocytes may be crucial for designing innovative MSC-based strategies in conditions in which B cells play a role, including systemic lupus erythematosus (SLE) and rejection of kidney transplantation. In this study, we show that, both in healthy subjects and in patients, in vitro B-cell proliferation, plasma-cell differentiation, and antibody production are inhibited by BM-derived MSCs when peripheral blood lymphocytes are stimulated with CpG, but not when sorted B cells are cultured with MSCs+CpG. Inhibition is restored in CpG+MSC cocultures when sorted T cells are added to sorted B cells, suggesting that this effect is mediated by T cells, with both CD4(+) and CD8(+) cells playing a role. Moreover, cell-cell contact between MSCs and T cells, but not between MSCs and B cells, is necessary to inhibit B-cell proliferation. Thus, the presence of functional T cells, as well as cell-cell contact between MSCs and T cells, are crucial for B-cell inhibition. This information can be relevant for implementing MSC-based therapeutic immune modulation in patients in whom T-cell function is impaired.

Use of rituximab in focal glomerulosclerosis relapses after renal transplantation.

Background. Focal and segmental glomerulosclerosis (FSGS) accounts for more than 10% of all cases of renal diseases leading to renal failure in children. After renal transplantation, 20% to 40% of FSGS relapse, frequently leading to renal loss. Plasmapheresis is considered the first option to treat relapses by several authors but is often ineffective. The anti-CD20 monoclonal antibody rituximab has been proposed as a possible treatment. Methods. We reviewed the effect of rituximab in seven children or young adults with pretransplant FSGS and relapse of proteinuria after transplantation who did not respond to intensive plasmapheresis. Results. After treatment, urine protein disappeared in three patients, was reduced by 70% in one patient and by 50% in one patient. No response was observed in one patient who had a quick deterioration of renal function and reached end-stage renal failure after 3 months. One additional patient developed a severe reaction a few minutes after the start of the first rituximab infusion. Conclusion. Rituximab is a possible option to treat some resistant cases of FSGS with relapsing proteinuria after transplantation. It is important that therapy is started early after evidence of failure of plasmapheresis, before sclerosis develops in the glomeruli. The response to treatment can occur after several months. During the follow-up period, CD19 cells should be monitored carefully, and additional rituximab infusions considered to maintain B-cell depletion.

Molecular analysis of the cystinuria disease gene: identification of four new mutations, one large deletion, and one polymorphism

Abstract A cystinuria disease gene (rBAT) has recently been identified, but evidence strongly suggests that only Type-I cystinuria is due to mutations in this gene. Sixteen point mutations and a large deletion causing the disease have so far been described in the rBAT gene sequence. To identify new mutated alleles, genomic DNA was analyzed, after the determination of the entire genomic structure of the rBAT gene, by RNA-single strand conformation polymorphism analysis, an accurate and sensitive method able to detect nucleotide changes. Four new point mutations, a large deletion, and a common intragenic polymorphism were detected. These new mutations increase to 22 the number of mutated alleles so far characterized in rBAT. In addition, the frequency of 21 mutations was assessed in a sample of accurately defined Type-I cystinuria choromosomes. They account for about 58% of all Type-I chromosomes, mutation M467T being the most common (0.26).

Simultaneous determination of inulin and p-aminohippuric acid in plasma and urine by reversed-phase high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method with UV detection was carried out to measure simultaneously plasma and urine concentrations of both p-aminohippuric acid and inulin. Following a simplified acid hydrolysis of the sample, the separation was carried out in 4 min using a C18 reversed-phase column with a flow-rate of 1 ml/min, and monitoring the absorbance at 280 nm. Within the investigated concentration ranges of inulin (0.1-3.2 mg/ml) and p-aminohippuric acid (0.0097-0.3 mg/ml), good linearity (r>0.99) was obtained. Within-run RSD ranged from 2.9 to 6.1% and between-run RSD ranged from 6.4 to 10%. Analytical recoveries were 101-112%, with little differences between plasma and urine samples. The detection limit was 1 microg/ml for all the analytes studied. This method might be ideal for renal function studies where a rapid and reproducible assessment of both renal glomerular filtration rate and blood flow-rate is required.

Longitudinal evaluation of mycophenolic acid pharmacokinetics in pediatric kidney transplant recipients. The role of post-transplant clinical and therapeutic variables

Abstract:  This longitudinal study assessed the influence of post‐transplant clinical and therapeutic variables in 50 kidney transplant recipients aged 2–19 yr receiving a triple immunosuppressive regimen consisting of cyclosporine microemulsion (CsA), steroids and MMF (300–400 mg/m2 body surface area twice daily), the full pharmacokinetic profile (10 points) of which was investigated on post‐transplant days 6, 30, 180 and 360. Total plasma MPA was measured by Enzyme Multiplied Immunoassay Technique. CsA therapeutic drug monitoring (TDM) was performed via C2 blood monitoring, while MPA TDM via C0. MPA Cmax, tmax, AUC0‐12 and AUC0‐4 pharmacokinetic profile changed significantly during the first post‐transplant year. C0 was a poor predictor of the total MPA exposure [as measured by the area under the concentration‐time curve AUC)], while a truncated AUC was a good surrogate of the 12‐h profile (r = 0.91; p < 0.001) Graft function and cyclosporine therapy influenced MPA pharmacokinetics, as shown by the univariate and multivariate analyses. We conclude that because after transplantation MPA exposure varied over time, a strict TDM is advisable in the pediatric population.

Three-yr safety and efficacy of everolimus and low-dose cyclosporine in de novo pediatric kidney transplant patients

The three yr results of a multicenter trial in de novo pediatric KT treated with a proliferative signal inhibitor and low dose CNI are presented. Thirty‐seven children (9.1 ± 5 yr old) received basiliximab, cyclosporine A (CyA C2:1400 ng/mL), (MMF C0:1.5–3 μg/mL), and prednisone. Three wk later everolimus was started (C0:5–10 ng/mL), CyA was reduced (C2:600 ng/mL after 90 days 300 ng/mL), and MMF discontinued. During the three‐yr period patient and graft survivals were 96%. One patient died for causes unrelated to the immunosuppression. Cumulative acute rejection rate including protocol and indication biopsies was 21.9%. None of the patients had signs of chronic humoral rejection. Incidence of dnDSA was 5%, 11%, and 22% at one, two, and three yr post‐transplant, respectively. Mean glomerular filtration rate measured at one yr and three yr post‐transplant was 105.5 ± 31 and 110.7 ± 27 mL/min/1.73 m2, respectively. A growth velocity of 7.7 ± 6.7 cm/yr was achieved with positive catch‐up growth. No malignancy or post‐transplant lymphoproliferative diseases were diagnosed. In conclusion, the treatment based on basiliximab induction, everolimus, low‐dose cyclosporine, and low‐dose prednisone leads to good long‐term efficacy in de novo pediatric KT recipients.

Post-transplant recurrence of atypical hemolytic uremic syndrome in a patient with thrombomodulin mutation

HUS is characterized by hemolytic anemia, thrombocytopenia, and acute renal failure. While “typical” HUS is usually associated with Shiga toxin‐producing Escherichia coli infections and recovers in the majority of cases, aHUS is caused by mutations of complement components or antibodies against CFH leading to uncontrolled activation of alternative complement pathway and often to ESRD. Recently, THBD gene mutations have been reported in aHUS. Theoretically, the risk of disease recurrence after renal transplantation should be low because THBD is primarily a membrane‐bound protein expressed by endothelial cells; however, a small proportion of THBD is present as a soluble form in plasma. We report the case of a 19‐yr‐old man with aHUS secondary to a THBD mutation that relapsed twice after two renal transplantations performed 12 yr apart. Despite successful control of HUS with plasma exchange and eculizumab after the second transplantation, the graft was ultimately lost due to severe steroid‐resistant cellular rejection. The present report suggests that THBD mutations may favor‐relapse of aHUS after renal transplantation.

Cytomegalovirus Infection in Pediatric Renal Transplantation and the Impact of Chemoprophylaxis With (Val-)Ganciclovir

Background Cytomegalovirus (CMV) replication and disease, with its associated morbidity and poor transplant outcome, represents a serious threat to transplant recipients. The pediatric kidney transplant population is at a particularly increased risk of CMV infection. Methods We therefore analyzed CMV epidemiology in a large cohort of pediatric renal transplant recipients (n = 242) and assessed the impact of antiviral chemoprophylaxis with valganciclovir (VGCV) or ganciclovir (GCV) on CMV replication and morbidity. Results While antiviral chemoprophylaxis with VGCV or GCV in patients with a high (D+/R−) or intermediate (D+/R+) CMV risk (n = 82) compared to preemptive therapy (n = 47) had no significant effect on the incidence of CMV syndrome or tissue-invasive disease, chemoprophylaxis was associated with a better preservation of transplant function at 3 years posttransplant (loss of estimated glomerular filtration rate in the chemoprophylaxis cohort, 16.0 ± 3.4 vs. 30.1 ± 4.7 mL/min per 1.73 m2 in the preemptive therapy cohort, P < 0.05).CMV replication was associated with a more pronounced decline of graft function (difference in estimated glomerular filtration rate of 9.6 mL/min per 1.73 m2 at 3 years) compared to patients without CMV replication. However, patients undergoing VGCV or GCV chemoprophylaxis had more leukocytopenia. Conclusion Antiviral chemoprophylaxis with VGCV or GCV in recipients with a high or moderate CMV risk is associated with a better preservation of transplant function. Hence, the prevention of CMV replication in this patient population has the potential to improve transplant outcome.