Complete steroid avoidance is effective and safe in children with renal transplants: a multicenter randomized trial with three-year follow-up.

To determine whether steroid avoidance in pediatric kidney transplantation is safe and efficacious, a randomized, multicenter trial was performed in 12 pediatric kidney transplant centers. One hundred thirty children receiving primary kidney transplants were randomized to steroid-free (SF) or steroid-based (SB) immunosuppression, with concomitant tacrolimus, mycophenolate and standard dose daclizumab (SB group) or extended dose daclizumab (SF group). Follow-up was 3 years posttransplant. Standardized height Z-score change after 3 years follow-up was -0.99 ± 2.20 in SF versus -0.93 ± 1.11 in SB; p = 0.825. In subgroup analysis, recipients under 5 years of age showed improved linear growth with SF compared to SB treatment (change in standardized height Z-score at 3 years -0.43 ± 1.15 vs. -1.07 ± 1.14; p = 0.019). There were no differences in the rates of biopsy-proven acute rejection at 3 years after transplantation (16.7% in SF vs. 17.1% in SB; p = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the SF and 90% in the SB arms (p = 0.30) at 3 years follow-up. Over the 3 year follow-up period, the SF group showed lower systolic BP (p = 0.017) and lower cholesterol levels (p = 0.034). In conclusion, complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants.

A peripheral blood diagnostic test for acute rejection in renal transplantation.

Monitoring of renal graft status through peripheral blood (PB) rather than invasive biopsy is important as it will lessen the risk of infection and other stresses, while reducing the costs of rejection diagnosis. Blood gene biomarker panels were discovered by microarrays at a single center and subsequently validated and cross-validated by QPCR in the NIH SNSO1 randomized study from 12 US pediatric transplant programs. A total of 367 unique human PB samples, each paired with a graft biopsy for centralized, blinded phenotype classification, were analyzed (115 acute rejection (AR), 180 stable and 72 other causes of graft injury). Of the differentially expressed genes by microarray, Q-PCR analysis of a five gene-set (DUSP1, PBEF1, PSEN1, MAPK9 and NKTR) classified AR with high accuracy. A logistic regression model was built on independent training-set (n = 47) and validated on independent test-set (n = 198)samples, discriminating AR from STA with 91% sensitivity and 94% specificity and AR from all other non-AR phenotypes with 91% sensitivity and 90% specificity. The 5-gene set can diagnose AR potentially avoiding the need for invasive renal biopsy. These data support the conduct of a prospective study to validate the clinical predictive utility of this diagnostic tool.

Identification of podocalyxin-like protein as a high endothelial venule ligand for L-selectin: parallels to CD34.

The leukocyte adhesion molecule, L-selectin, mediates the recruitment of lymphocytes to secondary lymphoid organs via interactions with specific ligands presented on high endothelial venules (HEV). Although the HEV-derived ligands for L-selectin are still incompletely defined, they share a common sialomucin-like structure which is thought to present clustered oligosaccharides to the lectin domain of L-selectin. Podocalyxin-like protein (PCLP) is a transmembrane sialomucin that is similar in structure to the well-characterized L-selectin ligand CD34. PCLP has been shown previously to be expressed on the foot processes of podocytes in the kidney glomerulus as well as on vascular endothelium at some sites. We have determined that PCLP is present on HEV, where it binds to both recombinant L-selectin and the HEV-specific monoclonal antibody MECA-79. Furthermore, purified HEV-derived PCLP is able to support the tethering and rolling of lymphocytes under physiological flow conditions in vitro. These results suggest a novel function for PCLP as an adhesion molecule and allow the definition of conserved structural features in PCLP and CD34, which may be important for L-selectin ligand function.

Anuria, omphalocele, and perinatal lethality in mice lacking the CD34-related protein podocalyxin.

Podocalyxin is a CD34-related sialomucin that is expressed at high levels by podocytes, and also by mesothelial cells, vascular endothelia, platelets, and hematopoietic stem cells. To elucidate the function of podocalyxin, we generated podocalyxin-deficient (podxl(-/)-) mice by homologous recombination. Null mice exhibit profound defects in kidney development and die within 24 hours of birth with anuric renal failure. Although podocytes are present in the glomeruli of the podxl(-/)- mice, they fail to form foot processes and slit diaphragms and instead exhibit cell--cell junctional complexes (tight and adherens junctions). The corresponding reduction in permeable, glomerular filtration surface area presumably leads to the observed block in urine production. In addition, podxl(-/)- mice frequently display herniation of the gut (omphalocele), suggesting that podocalyxin may be required for retraction of the gut from the umbilical cord during development. Hematopoietic and vascular endothelial cells develop normally in the podocalyxin-deficient mice, possibly through functional compensation by other sialomucins (such as CD34). Our results provide the first example of an essential role for a sialomucin in development and suggest that defects in podocalyxin could play a role in podocyte dysfunction in renal failure and omphalocele in humans.

A spacer protein in the Saccharomyces cerevisiae spindle poly body whose transcript is cell cycle-regulated.

Monoclonal antibodies against the 110-kD component of the yeast spindle pole body (SPB) were used to clone the corresponding gene SPC110. SPC110 is identical to NUF1 (Mirzayan, C., C. S. Copeland, and M. Synder. 1992. J. Cell Biol. 116:1319-1332). SPC110/NUF1 has an MluI cell cycle box consensus sequence in its putative promoter region, and we found that the transcript was cell cycle regulated in a similar way to other MluI-regulated transcripts. Spc110p/Nuflp has a long central region with a predicted coiled-coil structure. We expressed this region in Escherichia coli and showed by rotary shadowing that rods of the predicted length were present. The 110-kD component is localized in the SPB to the gap between the central plaque and the sealed ends of the nuclear microtubules near the inner plaque (Rout, M., and J. V. Kilmartin. 1990. J. Cell Biol. 111:1913-1927). We found that rodlike structures bridge this gap. When truncations of SPC110 with deletions in the coiled-coil region of the protein replaced the wild-type gene, the gap between the central plaque and the ends of the microtubules decreased in proportion to the size of the deletion. This suggests that Spc110p connects these two parts of the SPB together and that the coiled-coil domain acts as a spacer element.

Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate.

Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (PTPRO:) was disrupted at an exon coding for the NH(2)-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro(-/-) genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro(-/-) mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro(-/-) mice. There was no detectable increase in the urine albumin excretion of Ptpro(-/-) mice. After removal of one or more kidneys, Ptpro(-/-) mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

Hemodialysis followed by continuous hemofiltration for treatment of lithium intoxication in children.

Hemodialysis is the usual recommended treatment for severe lithium intoxication; however, rebound of lithium levels may require repeated hemodialysis treatments. We proposed that the addition of continuous hemofiltration after hemodialysis would prevent rebound by providing ongoing clearance of lithium. We report two pediatric patients with lithium intoxication treated by hemodialysis followed by continuous venovenous hemofiltration with dialysis (CVVHD). Both patients were symptomatic at presentation and had initial lithium levels more than three times the usual therapeutic range. Hemodialysis followed by CVVHD resulted in rapid resolution of symptoms, followed by continuous clearance of lithium without requiring repeated hemodialysis sessions. Both patients had return of normal mental status during CVVHD treatment, and neither patient experienced complications of hemodialysis or CVVHD. Total duration of treatment with hemodialysis followed by CVVHD was 34.5 hours for the first patient and 26 hours for the second patient. We conclude that hemodialysis followed by CVVHD is a safe and effective approach to the management of lithium intoxication in children.

Microplate-reverse hybridization method to determine dengue virus serotype.

A reverse transcriptase-polymerase chain reaction (RT-PCR) and microplate-reverse hybridization method were developed to detect and type dengue viruses in patients plasma specimens. A silica method was used to isolate RNA; and 3'-noncoding region universal primers were used to amplify dengue virus RNA. Using RT-PCR and ethidium bromide staining we could detect dengue virus in serum spiked with serially diluted dengue virus with a level of sensitivity similar to that of a quantitative fluorescent focus assay of dengue viruses in cell culture, i.e. 1.4 fluorescent focus units per reaction. Applying this assay to 14 dengue-positive plasma samples and 13 dengue-negative samples, dengue viremia was detectable by RT-PCR with a sensitivity comparable to mosquito inoculation. To determine the serotypes, digoxigenin-labeled PCR products from plasma samples and six laboratory adapted dengue viruses were hybridized in stringent conditions to serotype-specific DNA probes immobilized on microplates, and the hybridized product was detected with a colorimetric assay. Serotypes of dengue viruses, in cell culture and in patient plasma specimens, were identified using this method.

Peritoneal dialysis for management of pediatric acute renal failure.

BACKGROUND: While the use of continuous renal replacement therapies in the management of children with acute renal failure (ARF) has increased, the role of peritoneal dialysis (PD) in the treatment of pediatric ARF has received less attention. DESIGN: Retrospective database review of children requiring PD for ARF over a 10-year period. SETTING: Pediatric intensive care unit at a tertiary-care referral center. PATIENTS: Sixty-three children without previously known underlying renal disease who required PD for treatment of ARF. RESULTS: Causes of ARF were congestive heart failure (27), hemolytic-uremic syndrome (13), sepsis (10), nonrenal organ transplant (7), malignancy (3), and other (3). Mean duration of PD was 11 +/- 13 days. Children with ARF were younger (30 +/- 48 months vs 88 +/- 68 months old, p < 0.0001) and smaller (11.9 +/- 15.9 kg vs 28 +/- 22 kg, p < 0.0001) than children with known underlying renal disease who began PD during the same time period. Percutaneously placed PD catheters were used in 62% of children with ARF, compared to 4% of children with known renal disease (p < 0.0001). Hypotension was common in patients with ARF (46%), which correlated with a high frequency of vasopressor use (78%) at the time of initiation of PD. Complications of PD occurred in 25% of patients, the most common being catheter malfunction. Recovery of renal function occurred in 38% of patients; patient survival was 51%. CONCLUSIONS: Peritoneal dialysis remains an appropriate therapy for pediatric ARF from many causes, even in severely ill children requiring vasopressor support. Such children can be cared for without the use of more expensive and technology-dependent forms of renal replacement therapies.

Pediatric primary care provider's knowledge of HIV/AIDS care.

To respond to the difficulties that community-based providers face in keeping abreast of the rapid changes in HIV-related care, an intensive pediatric HIV mentoring program (Pediatric HIV Miniresidency [MR]) was developed, linking a regional AIDS Education and Training Center (AETC) with an urban children's hospital HIV outpatient care site. The purpose of this study was to evaluate HIV-related knowledge and perceived skills, abilities, and willingness of community-based primary care pediatric providers and providers completing the MR. A convenience sample of community-based primary pediatric practitioners and those participants in the MR program completed a three-part mailed survey. The survey assessed practice characteristics, knowledge of pediatric HIV clinical care, and perceived skills, ability, and willingness (PSAW) to provide HIV-related care. The main outcome measures were overall knowledge and PSAW scores. One hundred nineteen community-based practitioners (NMRs), 20% of those surveyed, completed the instrument, as did 19 of 20 MR participants. NMRs exhibited low knowledge scores in key areas relating to the identification and evaluation of HIV-exposed children. Fewer than half of these respondents correctly answered questions related to HIV antibody incidence in HIV-exposed newborns and recommended diagnostic testing of such infants. Providers completing the MR scored significantly higher on the knowledge survey (15.2 vs. 8.8, p < 0.001), and had higher PSAW scores (45.8 vs. 33.9, p < 0.001). Although the generalizability of our study is limited by the low response rate, community-based physicians completing the survey demonstrated a lack of knowledge we believe necessary to provide pediatric HIV-related care (as defined by Public Health Service practice guidelines). Physicians completing the MR program had substantial HIV-related knowledge and expressed a willingness to provide care to HIV-exposed/infected children. An effective MR program provides a mechanism for developing a network of dedicated community-based physicians who are willing and capable of providing care to HIV-infected or exposed infants and children.

Response to early measles-mumps-rubella vaccination in infants with chronic renal failure and/or receiving peritoneal dialysis.

Achieving immunity to childhood viral infections before renal transplantation is crucial. However, children with chronic renal failure (CRF) may respond poorly to vaccination, making it difficult to achieve immunity before transplantation, particularly if they will require transplantation at a young age. To address this problem, we developed a protocol of early measles-mumps-rubella (MMR) vaccination in infants with CRF. Nine infants received MMR vaccine at a mean age of 11.6 +/- 2.5 months. When vaccinated, 6 of the children (67%) were on peritoneal dialysis, and 3 (33%) had CRF [glomerular filtration rate (GFR) < 30 mL/min/1.73 m2]. Eight patients were later transplanted at a mean age of 16.8 +/- 4.8 months. Titers were measured before transplantation in all patients. Response to vaccination was excellent, with 89% developing immunity to measles, 88% developing immunity to mumps, 100% developing immunity to rubella, and 88% developing immunity to all three components of the vaccine. These response rates were equivalent to, or slightly better than, those previously reported by Schulman for older children (19 +/- 6 months) on dialysis: 80% for measles, 50% for mumps, 100% for rubella, and 30% for all three components. We conclude that early MMR vaccination induces immunity in most infants with CRF, even those on peritoneal dialysis. Response rates are similar to those previously reported in older children. This approach may help to facilitate transplantation in young infants by achieving immunity earlier than traditional vaccination schedules.

Improvement of glycemic control by CAPD with intraperitoneal insulin in a child with IDDM and ESRD.

Use of intraperitoneal insulin in diabetic end-stage renal disease (ESRD) patients receiving continuous ambulatory peritoneal dialysis (CAPD) is known to result in improved glycemic control. This route of insulin administration, although standard in adult diabetic CAPD patients, has not previously been reported in children. A 12-year old boy with ESRD from renal dysplasia who also had insulin-dependent diabetes mellitus (IDDM) was treated with CAPD and intraperitoneal insulin prior to renal transplantation. Diabetes and renal dysplasia were both diagnosed at 11 weeks of age. When he reached end-stage he was initially started on hemodialysis via a central line but was switched to CAPD because of recurrent line sepsis. His IDDM had been poorly controlled up to that time. CAPD was performed using 4 exchanges per day of 1.5% dialysate with a fixed dose of insulin added to each bag and with adjustments made based on blood glucose. His glycemic control markedly improved, with a fall in his glycosylated hemoglobin from 13.6% to 6%. CAPD was continued for 7 months until a living-related renal transplant was performed. Two episodes of peritonitis occurred while the patient received CAPD (1 episode/3.5 patient-months). We conclude that the use of intraperitoneal insulin in children with IDDM and ESRD leads to improved glycemic control. The rate of peritonitis, however, may be increased in these children.

Pulmonary function variation in ventilator dependent critically ill infants on peritoneal dialysis.

Four ventilator dependent infants on PD for acute renal failure underwent pulmonary function (PFT) evaluation at varying times in the PD cycle. Mid dwell peak intraperitoneal pressure (IPP) correlated with a significant decrease in pulmonary compliance and increase in air way resistance. This further correlated with a decrease in PO2 and an increase in PCO2 on arterial blood gas analysis. Etiology of the PFT changes appear to correlate most closely with IPP yet other factors including pulmonary artery shunting as well as hypercapnea secondary to a 4.25% dialysate are being evaluated as additional causative factors.