Jacek Zachwieja

Cranioectodermal Dysplasia, Sensenbrenner Syndrome, Is a Ciliopathy Caused by Mutations in the IFT122 Gene

Cranioectodermal dysplasia (CED) is a disorder characterized by craniofacial, skeletal, and ectodermal abnormalities. Most cases reported to date are sporadic, but a few familial cases support an autosomal-recessive inheritance pattern. Aiming at the elucidation of the genetic basis of CED, we collected 13 patients with CED symptoms from 12 independent families. In one family with consanguineous parents two siblings were affected, permitting linkage analysis and homozygosity mapping. This revealed a single region of homozygosity with a significant LOD score (3.57) on chromosome 3q21-3q24. By sequencing candidate genes from this interval we found a homozygous missense mutation in the IFT122 (WDR10) gene that cosegregated with the disease. Examination of IFT122 in our patient cohort revealed one additional homozygous missense change in the patient from a second consanguineous family. In addition, we found compound heterozygosity for a donor splice-site change and a missense change in one sporadic patient. All mutations were absent in 340 control chromosomes. Because IFT122 plays an important role in the assembly and maintenance of eukaryotic cilia, we investigated patient fibroblasts and found significantly reduced frequency and length of primary cilia as compared to controls. Furthermore, we transiently knocked down ift122 in zebrafish embryos and observed the typical phenotype found in other models of ciliopathies. Because not all of our patients harbored mutations in IFT122, CED seems to be genetically heterogeneous. Still, by identifying CED as a ciliary disorder, our study suggests that the causative mutations in the unresolved cases most likely affect primary cilia function too.

Increased apoptosis of peripheral blood lymphocytes in children with nephrotic syndrome.

Abstract Nephrotic syndrome is accompanied by and probably related to abnormal T-lymphocyte function. Decreased stimulation of survival factors and increased levels of ”dead signals” may lead to the malfunction of many cells, including lymphocytes. In our study, we investigated the process of apoptosis within T cells in children with a first attack of nephrotic syndrome (NS). We found that the number of apoptotic T cells is greater in these patients than in both children in remission from NS and in controls. The percentage of annexin-V-fluorescein isothiocyanate (FITC)-positive CD3+ cells was 27.30± 12.13% in children with a first attack of NS, 19.22± 15.16% (P=0.006) in children in remission and 16.20± 6.13% (P=0.004) in controls. The percentage of annexin-V-FITC-positive CD3+CD4+ cells was 7.35±7.72% in children with a first attack of NS, 3.80±3.75% (P=0.0001) in children in remission and 3.82±2.01% (P=0.0002) in controls. We conclude that abnormal number and function of T lymphocytes found in NS patients may be related to an increased apoptotic rate of circulating lymphocytes.

Familial, recurrent haemolytic-uraemic syndrome with hypocomplementaemia

We read with interest the report by Bogdanovic et al. [1] on haemolytic-uraemic syndrome (HUS) associated with Aeromonas hydrophila enterocolitis. Since July 1987, we have assessed 82 children with typical HUS. Stool specimens from each child with HUS were tested for the presence of multiple pathogens including verotoxin-producing Escherichia coli, Salmonella, Shigella, CampyIobacter, Yersinia and A. hydrophila. Oxidase testing was routinely performed on all stool isolates. Forty-five (55%) children had E. coli 0157 : H7, 2 (2%) children had A. hydrophila, and 1 child (1%) had Campylobacter in the stool. We concur with Bogdanovic et al. [1] thatA, hydrophila should be added to the list of bacteria that may trigger an episode of typical (D+) HUS and that oxidase testing should be routinely performed on all stool isolates in patients with HUS. Wm. Lane M. Robson 1 Alexander K. C. Leung 1 Cynthia L. Trevenen 2 Departments of I Pediatrics and 2 Pathology University of Calgary Alberta Childrens Hospital 300, 10601 Southport Road SW Calgary, Alberta T2W 3M6 Canada

Ezrin—a useful factor in the prognosis of nephrotic syndrome in children: an immunohistochemical approach

Background: Minimal change disease (MCD) and diffuse mesangial proliferation (DMP) are the most common pathomorphological forms of nephrotic syndrome glomerulopathies in children. The clinical course of DMP can be characterised by either DMP-sensitivity (DMP-S) or DMP-resistance (DMP-R) to steroids, resulting in an unfavourable course of the glomerulopathy. Although the clinical processes of DMP-S and DMP-R are initially identical, resistance to steroids may be foreseen by the immunohistochemical expression of cytoskeleton-associated proteins in podocytes. Aims: To estimate the immunohistochemical expression of ezrin in children with MCD, DMP and focal segmental glomerulosclerosis (FSGS) and to evaluate its usefulness in predicting resistance to steroids. Materials and methods: Renal biopsy specimens of patients with MCD (n = 15), DMP (n = 16) and FSGS (n = 6) were taken. The control tissue consisted of normal-appearing cortex taken from kidneys resected for localised neoplasms (n = 6). The indirect immunohistochemical protocol for the use of a monoclonal antibody directed against ezrin was used. Results: The immunohistochemical expression of ezrin in cases progressively reduced from MCD to DMP-S to DMP-R to FSGS. Except for DMP-R and FSGS (p>0.05), the difference in ezrin expression in podocytes was significant. Conclusion: Ezrin can be a potent marker of podocyte injury (podocytopathy) and may help in the histological qualification of MCD, DMP and FSGS. The increased permeability of the filtration barrier in steroid-resistant and proteinuric glomerulopathies may be a consequence of subcellular changes in podocyte-associated proteins following decreased expression of ezrin.

Abnormal cytokine synthesis as a consequence of increased intracellular oxidative stress in children treated with dialysis.

Background/Aim: End-stage renal disease (ESRD) induces a clinical state of immunodeficiency with a higher incidence of infections and higher mortality due to infectious complications compared with the normal population. The definite mechanism responsible for the host defense alterations is not well understood. The aim of the study was to investigate intracellularly the relationship between cytokine synthesis and oxidative stress in peripheral blood lymphocytes in children with ESRD. Methods: Twenty-one children (age 11.7 ± 5.8 years) with ESRD treated with hemodialysis (HD; n = 10) and peritoneal dialysis (PD; n = 11) were studied. Nine healthy children of comparable age formed the control group. To determine intracellular oxidative stress we used dihydrorhodamine-123 (DHR), which after oxidation to rhodamine-123 (RHO) emitted a bright fluorescent signal. Intracellular oxidation of DHR in T lymphocytes reflected intracellular oxidative stress. The intracellular synthesis of cytokines (IL-2, IFN-γ, IL-4, IL-6) was also measured. Both parameters were detected at a single-cell level by flow cytometry. Lymphocyte subsets were evaluated using the monoclonal antibodies conjugated with fluorochromes. Results: We found that in T lymphocytes the mean fluorescence intensity (MFI), which reflected intracellular oxidative stress, was increased in ESRD patients compared to the controls (CD3+: 34.77 ± 11.55 vs. 22.55 ± 4.97, p < 0.01; CD3+CD8+: 34.31 ± 12.17 vs. 20.77 ± 4.89, p < 0.01; CD3+CD4+: 36.06 ± 6.98 vs. 24.44 ± 7.68, p < 0.001). HD patients showed slightly higher MFI compared to PD patients in CD3+ cells (39.32 ± 11.70 vs. 30.63 ± 10.20, NS), in CD3+CD8+ cells (37.90 ± 14.32 vs. 31.06 ± 9.34, NS) and in CD3+CD4+ cells (40.10 ± 2.28 vs. 29.33 ± 7.06, p < 0.001). The intracellular synthesis of IL-2 was higher in ESRD patients compared to the controls, both in CD3+ cells (31.34 ± 9.80 vs. 20.49 ± 15.26%, p < 0.05) and in CD3+CD4+ cells (36.10 ± 8.69 vs. 24.03 ± 16.95%, p < 0.05). The intracellular synthesis of IFN-γ, IL-4 and IL-6 was significantly lower in the ESRD group compared to the controls. Interestingly, in patients treated with HD, negative correlations between the degree of intracellular oxidative stress and intracellular cytokine synthesis in CD3+ lymphocytes were found. Conclusion: Our results show that patients with ESRD, especially those treated with HD, present increased oxidative stress in T lymphocytes, which may lead to decreased cytokine synthesis and abnormal immune response.

Mycophenolate mofetil (MMF) treatment efficacy in children with primary and secondary glomerulonephritis

Introduction The aim of our study was to analyse the efficacy and safety of mycophenolate mofetil (MMF) as part of the complex immunosuppressive therapy in children with different types of primary and secondary glomerulonephritis, who were not eligible for the standard treatment routine suggested by evidence-based guidelines. Material and methods The study group comprised 85 children with proteinuric glomerulopathies hospitalized between 2007 and 2010, who were non-responders to immunosuppressive therapy. The dose of MMF was established as 1 g/m2/24 h. Remission was defined as negative proteinuria in three consecutive urinalyses. Results The patients were divided into 4 groups: idiopathic nephrotic syndrome (n = 35), primary glomerulonephritis (n = 15), auto-antibody associated glomerulonephritis (n = 20) and lupus nephropathy (LN, n = 15). Ten patients from the first group (29%) and 5 patients each from the second and third group (34% and 25% respectively) did not respond to MMF therapy. On the other hand, all the children diagnosed with LN have reached clinical and biochemical remission. Conclusions Alternative rescue MMF therapy should always be taken into consideration in proteinuric patients who are non-responders to steroids, cyclosporine A and cyclophosphamide in whom the initial glomerular filtration rate is higher than 60 ml/min/1.73m2. It is recommended that MMF be administered as part of the standard treatment regimen in patients diagnosed with lupus nephropathy. In these groups of patients, the potent benefits of this therapy are higher than expected side-effects.

Monitoring of mycophenolate mofetil metabolites in children with nephrotic syndrome and the proposed novel target values of pharmacokinetic parameters

The aim of the study was to estimate target values of mycophenolate mofetil (MMF) pharmacokinetic parameters in children with proteinuric glomerulopathies by calculating the pharmacokinetic parameters of MMF metabolites (mycophenolic acid [MPA], free MPA [fMPA] and MPA glucuronide [MPAG]) and assessing their relation to proteinuria recurrence. One hundred and sixty-eight blood samples were collected from children, aged 3-18 years, diagnosed with nephrotic syndrome or lupus nephritis. MMF metabolites concentrations were examined before drug administration (Ctrough) and up to 12h afterward employing high-performance liquid chromatography. Dose-normalized MPA Ctrough and area under the concentration-time curve from 0 to 12h (AUC12) were within 0.29-6.47 μg/mL/600 mg/m(2) and 9.97-105.52 μg h/mL/600 mg/m(2), respectively. MPA Ctrough was twofold lower (p=0.024) in children with proteinuria recurrence. MPA, fMPA and MPAG concentrations correlated positively to respective AUC12. It may be suggested MMF metabolites monitoring in children with proteinuric glomerulopathies is justified by MPA Ctrough<2 μg/mL in patients at risk of the proteinuria recurrence. Such a recurrence is most probably caused by not sufficient MPA concentration during proteinuric glomerulopathies treatment. MPA Ctrough>3 μg/mL may be considered as an efficient one to avoid proteinuria recurrence. Finally, MPA target AUC12 should exceed 60 μg h/mL to ensure the safe and effective treatment in children with nephrotic syndrome, however, the upper limit is still to be established.

A prospective study to determine the significance of ventricular late potentials in children with mitral valvar prolapse

We aimed prospectively to determine the incidence of ventricular arrhythmias and ventricular late potentials in children with mitral valvar prolapse, and to assess whether signal-averaged electrocardiography could identify which such children were at high risk of developing ventricular tachycardia. In all, we examined 151 children with mitral valvar prolapse, at an age of 12.2 +/- 3.1 years, and 164 healthy subjects aged 12.3 +/- 3.7 years. All children underwent 24-hour ambulatory Holter monitoring and echocardiography. The children with mitral valvar prolapse were followed prospectively for a mean of 64 months. There was a significantly higher prevalence of ventricular arrhythmias in those with prolapse than in the controls (p < 0.0001). Runs of ventricular tachycardia were observed in 3 children with mitral valvar prolapse compared with one from the control group. Late potentials were more frequently observed in the children with mitral valvar prolapse than in those who were healthy (p < 0.0001), and also in those with prolapse suffering ventricular arrhythmias compared with those without ventricular arrhythmias (p < 0.02). During follow-up, 24 children with prolapsing mitral valves developed non-sustained ventricular tachycardia, giving a frequency of 3.1/100 subject-years. The sensitivity of late potentials was low, at 52%, for the identification of children with mitral valvar prolapse who developed ventricular tachycardia, although the specificity was high at 90%. This gave a positive predictive value of 50%, and a negative predictive value of 91%. We conclude that prolapse of the mitral valve predisposes to the development of ventricular arrhythmias and late potentials in children. An abnormal signal-averaged electrocardiogram is a specific, but not very sensitive, predictor for the development of ventricular tachycardia in such children.

An Evaluation of the Efficacy of Selective Alpha-Blockers in the Treatment of Children with Neurogenic Bladder Dysfunction--Preliminary Findings.

The aim of this study was to assess the usefulness of selective α1-blockers in children with neurogenic urinary tract dysfunctions and increased leak point pressure (LPP). 14 children from age 6 to 16 years with neurogenic urinary tract dysfunctions (neurogenic bladder) and LPP > 40 cm H2O were enrolled in the study. All patients received a selective α1-blocker (doxazosin) for 6–8 weeks with an initial dosage of 0.03 mg/kg. During the observation period the continuation of oral anticholinergics, Clean Intermittent Catheterization (CIC), observation of “urinary dryness” and urinary incontinence periods were recommended. Patients were scheduled for a follow-up visit and urodynamic investigation after 6–8 weeks after the doxazosin therapy was started. In 4 patients, urine leakage occurred at lower pressures; in 9 patients, no significant changes in urine leak point pressures were detected; in 3 patients, there was a significant increase in the bladder capacity; in one patient, deterioration in continence was noted. The differences both in LPP and LPV before and after the treatment were not statistically significant. Our observations are consistent with the conclusions from other studies and showed no evident efficacy of doxazosin in children with neurogenic bladder.

Efficacy and safety of rituximab treatment in children with primary glomerulonephritis.

BACKGROUND The aim of our study was to analyze the efficacy and safety of rituximab, a chimeric monoclonal antibody against CD20 lymphocytes, as a nonstandard immunosuppressive therapy in children with different types of primary glomerulonephritis who were not eligible for routine treatment. METHODS The study group was composed of 16 children with proteinuric glomerulopathies, not responding to standard immunosuppressive therapy. The indications included steroid-resistant nephrotic syndrome (n=14) and steroid-dependent nephrotic syndrome (n=2). The dose of rituximab was established as 375 mg/m2 of body surface area, administered by intravenous infusion once weekly for 1 to 4 weeks, depending on the CD19 lymphocyte count. We evaluated proteinuria and plasma concentration of CD19 lymphocytes at intervals of 1, 3 and 6 months, after which patients received a single repeat dose. RESULTS Remission, defined as proteinuria less than 150 mg per 24 hours, was observed in 7 of the 16 children. There were no statistically significant differences in leukocyte counts between single and multiple rituximab doses. We also did not observe any clinical or biochemical side effects. CONCLUSIONS In conclusion, we postulate that alternative rituximab therapy should be taken into consideration in nephrotic patients not responding to standard therapy.