Update of PAX2 mutations in renal coloboma syndrome and establishment of a locus-specific database.

Renal coloboma syndrome, also known as papillorenal syndrome is an autosomal-dominant disorder characterized by ocular and renal malformations. Mutations in the paired-box gene, PAX2, have been identified in approximately half of individuals with classic findings of renal hypoplasia/dysplasia and abnormalities of the optic nerve. Prior to 2011, there was no actively maintained locus-specific database (LSDB) cataloguing the extent of genetic variation in the PAX2 gene and phenotypic variation in individuals with renal coloboma syndrome. Review of published cases and the collective diagnostic experience of three laboratories in the United States, France, and New Zealand identified 55 unique mutations in 173 individuals from 86 families. The three clinical laboratories participating in this collaboration contributed 28 novel variations in 68 individuals in 33 families, which represent a 50% increase in the number of variations, patients, and families published in the medical literature. An LSDB was created using the Leiden Open Variation Database platform: www.lovd.nl/PAX2. The most common findings reported in this series were abnormal renal structure or function (92% of individuals), ophthalmological abnormalities (77% of individuals), and hearing loss (7% of individuals). Additional clinical findings and genetic counseling implications are discussed.

Inhibition of Simian Virus 40 replication by targeting the molecular chaperone function and ATPase activity of T antigen.

Polyomaviruses such as BK virus and JC virus have been linked to several diseases, but treatments that thwart their propagation are limited in part because of slow growth and cumbersome culturing conditions. In contrast, the replication of one member of this family, Simian Virus 40 (SV40), is robust and has been well-characterized. SV40 replication requires two domains within the viral-encoded large tumor antigen (TAg): The ATPase domain and the N-terminal J domain, which stimulates the ATPase activity of the Hsp70 chaperone. To assess whether inhibitors of polyomavirus replication could be identified, we examined a recently described library of small molecules, some of which inhibit chaperone function. One compound, MAL2-11B, inhibited both TAg's endogenous ATPase activity and the TAg-mediated activation of Hsp70. MAL2-11B also reduced SV40 propagation in plaque assays and compromised DNA replication in cell culture and in vitro. Furthermore, the compound significantly reduced the growth of BK virus in a human kidney cell line. These data indicate that pharmacological inhibition of TAg's chaperone and ATPase activities may provide a route to combat polyomavirus-mediated disease.

Loop-mediated isothermal amplification assay for rapid detection of common strains of Escherichia coli.

We developed a highly sensitive and specific LAMP assay for Escherichia coli. It does not require DNA extraction and can detect as few as 10 copies. It detected all 36 of 36 E. coli isolates and all 22 urine samples (out of 89 samples tested) that had E. coli. This assay is rapid, low in cost, and simple to perform.

Renal transplantation in children managed with lymphocyte depleting agents and low-dose maintenance tacrolimus monotherapy.

OBJECTIVE: Describe the safety and efficacy of antithymocyte globulin or alemtuzumab preconditioning, steroid avoidance and reduced calcineurin inhibitor (CNI) immunosuppression in 34 children undergoing renal transplantation. METHODS: ATG (n=8) or alemtuzumab (n=26) were infused at the time of transplantation. This was followed by low-dose twice a day tacrolimus monotherapy with consolidation to once daily dosing by 6 months and once every other day dosing by 12 months. Follow-up ranged from 0.5-2.9 years (mean 1.33 years), with a minimum of 6 months. RESULTS: Both ATG and alemtuzumab were well tolerated. Lymphopenia occurred routinely and resolved after 3-6 months. Acute cellular rejection occurred in 9%; it was related to medical nonadherence in two patients and resulted in one graft loss at 1.5 years. Important adverse events included transient neutropenia in 10 children (none with serious infection), and autoimmune hemolytic anemia in two (resolved with a steroid course in both and conversion to sirolimus in one). Estimated glomerular filtration rate (e-GFR) was stable and averaged 88 mL/min/1.73 m2 at latest follow-up. Fifteen preadolescents had a greater increase in height Z-score at 1 year (1.3 vs. 0.5, P=0.001), and a higher e-GFR (94.8+/-21 vs. 76.6+/-20 ml/min/1.73 m2, P<0.05), when compared to case-matched historical controls who were weaned off steroids by 6 months after transplantation and received twice daily tacrolimus monotherapy. CONCLUSION: This simple regimen appears safe, has a low risk for acute cellular rejection or other adverse effects, and is associated with excellent growth and renal function. Such a regimen may also improve compliance and limit CNI nephrotoxicity.

Low incidence of BK virus nephropathy after simultaneous kidney pancreas transplantation.

BACKGROUND: BK virus renal allograft nephropathy (BKVAN) in the setting of simultaneous kidney-pancreas transplantation (SKPT) has been inadequately studied and reported. We analyzed our data on the incidence of BKVAN and its outcome in SKPT recipients at University of Pittsburgh Medical Center (UPMC) and affiliated centers and report significant differences compared to previous studies. METHODS: This study used retrospective review and case studies. RESULTS: A review of 243 consecutive SKPT recipients from January 1, 1996 to December 31, 2004 identified seven cases (three females; ages = 23-54 yrs) of BKVAN following SKPT (incidence = 2.9%). The immunosuppressive protocols during this period were divided into: Period I (pre-August 2001) with no antibody induction and Period II (post-August 2001) with alemtuzumab or antithymocyte globulin induction with steroid avoidance. One BKVAN case was diagnosed in Period II (incidence = 1.4%). Six of seven patients were treated with intravenous cidofovir (0.20-0.50 mg/kg) every two to four weeks over one to six months. Three patients lost the renal allograft 8-22 months following diagnosis of BKVAN, whereas four patients had prolonged allograft survival. Pancreatic function was well preserved in five; one patient lost the pancreatic function due to surgical complications and one has had partial preservation. CONCLUSIONS: There was a relatively lower incidence of BKVAN among SKPT patients at our center. Although overall graft loss rate was comparable to other series, BKVAN patients had a slightly prolonged graft life. The BKVAN incidence was further reduced in patients receiving modified immunosuppression with antibody preconditioning. The underlying reasons may include less toxic immunosuppressive protocols, earlier diagnosis and the use of antiviral therapy.

Detection of CD8+ T cells sensitized to BK virus large T antigen in healthy volunteers and kidney transplant recipients.

BK virus (BKV) infections after renal transplantation are increasingly recognized. Development of immune monitoring strategies against BKV requires definition of antigenic epitopes. Hence, T cells from HLA-A02-positive healthy subjects and kidney transplant recipients were stimulated by BKV lysate pulsed on mature autologous dendritic cells and screened against four different T antigen peptides or against BKV lysate. IFN-gamma production was measured by ELISPOT assays. The peptide BKV362-371 (MLTERFNHIL) was naturally processed and recognized by five of six healthy subjects (39 +/- 11 IFN-gamma spots/100,000 cells) and five of seven kidney transplant recipients (21 +/- 12 IFN-gamma spots). Less frequent and weaker CD8+ T-cell responses were detected against three other peptides. Thus, BKV large T antigen is a target for CD8+ T-cell immunity. T-antigen-specific T-cytotoxic cells circulate in healthy blood donors, implying that transient expression of T antigen presumably occurs at sites of viral latency and helps maintain a constant pool of circulating CD8+ T memory cells.

The pathobiology of polyomavirus infection in man.

This article traces the discovery of polyomaviruses and outlines investigations, which shed light on potential modes of transmission of this increasingly important group of human pathogens. The pathobiology of the virus is summarized with particular reference to interactions with host cell receptors, cell entry, cytoplasmic trafficking, and targeting of the viral genome to the nucleus. This is followed by a discussion of sites of viral latency and factors leading to viral reactivation. Finally, we present biochemical mechanisms that could potentially explain several key elements of tissue pathology characteristic of BKV mediated damage to human kidney.

Diagnosis and treatment of BK virus-associated transplant nephropathy.

The incidence of polyoma virus infection, particularly that of BK virus (BKV) in kidney transplant recipients has been increasing steadily since early 1990s. The diagnosis is generally made by a renal allograft biopsy. However the diagnosis can sometimes be difficult because of the pathological similarities between BKV associated nephropathy (BKVAN) and acute cellular rejection. In addition to the difficulties in making a diagnosis, the treatment of BKVAN can also be very complex. Reduction in immunosuppression is generally advocated as the initial therapeutic option for the management of BKVAN. Despite reduced immunosuppression, BKV can persist in the renal allograft and lead to gradual loss of kidney function. Hence, new therapeutic options are being evaluated for treatment of BKVAN. Cidofovir, an anti-viral agent with known nephrotoxic effects, has been successfully used in very low doses to treat patients with BKVAN, with serial measurement of the blood and urine BKV load with PCR assays. More recently several other agents have also been utilized to treat BKVAN, with variable success. This chapter summarizes the current diagnostic modalities and therapeutic options for BKVAN.

Monitoring for polyomavirus BK And JC in urine: comparison of quantitative polymerase chain reaction with urine cytology.

Clinical monitoring for polyomavirus infection is becoming common, but the optimal detection technique remains undefined. We compared the relative efficacy of exfoliative cytology and polymerase chain reaction (PCR) for detecting viruria in 100 urine samples. Definite decoy cells were seen in 8% and probable decoy cells in 5% of specimens. PCR showed BK virus (BKV) DNA in all these and in an additional 18% of samples. Using decoy cells as a marker of polyomavirus viruria cytology has a sensitivity of 41.9% and negative predictive value of 82.8%. The specificity and positive predictive value for viruria (not viral nephropathy) are 100%. False-negative results occurred in samples with suboptimal cellularity, vaginal contamination, and a low viral load. One patient with a false-negative urine cytology developed BKV nephropathy on follow-up. Compared with PCR, urine cytology is a less sensitive technique, which requires morphologically intact cells, and cannot distinguish BKV from JC virus.

Effect of leflunomide and cidofovir on replication of BK virus in an in vitro culture system.

BACKGROUND: Cidofovir and Leflunomide are used empirically in the treatment of BK virus nephropathy. The aim of this study is to quantify the antiviral activity of these drugs. METHODS: BK virus was grown in a cell-culture system. The rate of viral replication in the presence or absence of the drug being tested was assessed using a quantitative polymerase chain reaction assay. RESULTS: The inhibitory concentration, effective concentration, and selectivity index for Leflunomide are 39.7+/-6.9, 11.3+/-2.8, and 3.8+/-0.8 microg/mL, respectively. For Cidofovir, these indices were, respectively, 63.9+/-17.2, 36.3+/-11.7, and 2.3+/-0.8 microg/mL. CONCLUSIONS: The in vitro activity of Cidofovir and Leflunomide is modest, and the selectivity index is low. There is a need to develop more effective and less toxic anti-BK virus drugs for clinical use.

Quantitation of viral DNA in renal allograft tissue from patients with BK virus nephropathy.

BACKGROUND: BK virus (BKV) allograft nephropathy (BKVAN) is a complication in renal transplantation recipients. Histopathology is the gold standard for diagnosis. Quantitative polymerase chain reaction (PCR) assay for renal biopsy has not been evaluated as a diagnostic test. Determination of renal BKV load may identify patients at risk for disease before histologic nephropathy. METHODS: Quantitative PCR assay for BKV DNA was performed in 28 biopsies of patients with BKVAN; 50 biopsies were performed before a diagnosis of BKVAN, and 126 control biopsies were from patients without a history of BKVAN. RESULTS: BKV DNA was present in 19 of 50 (38%) biopsies performed 1 to 164 weeks before diagnosis of BKVAN. The viral load (mean 216 copies/cell) was lower than in biopsies of patients with BKVAN (mean 6063 viral copies/cell, <0.05). In 10 of 127 (7.8%) control biopsies, a low level of BKV DNA (mean 3.8 copies/cell) was found in three biopsies from chronic allograft nephropathy patients; two biopsies with acute rejection; four biopsies with borderline change; and one biopsy with cytomegalovirus nephritis. CONCLUSION: BKV load exceeding 59 copies per cell identified all cases of BKVAN. The diagnostic sensitivity, specificity, positive predictive value, and negative predictive value of quantitative PCR were 100%, 92.1%, 73.6%, and 100%, respectively. Lower levels of BKV DNA were identified in biopsies performed before viral nephropathy development. Future research will determine if earlier recognition of at-risk patients allows application of antiviral strategies to improve graft outcome.

DNA sequencing of viral capsid protein VP-1 region in patients with BK virus interstitial nephritis.

BACKGROUND: Mutations in the viral capsid protein VP-1 region are associated with increased pathogenicity of polyomavirus in experimental systems. This study sought to determine whether analogous viral genetic changes occur in human BK virus (BKV) interstitial nephritis (ISN). METHODS: PCR was used to amplify a 94-bp nucleotide sequence of the viral capsid protein VP-1 region (positions 1740-1833, Dun numbering) in 49 biopsies obtained from 24 patients with BKV-ISN. DNA sequencing was performed by the dideoxy method. RESULT: The VP-1 region was highly polymorphic and 22 "hot spots" of sequence variability were noted. Genotypes I, II, and IV were assigned to 13, 1, and 5 cases, respectively, but 5 cases could not be unambiguously classified due to sequence heterogeneity at sites used to discriminate between genotypes. Even in cases where genotypes could be assigned, only 5 biopsies showed complete sequence identity with published genotype sequences. Sequential biopsies showed temporal changes in one or more nucleotides in all patients with multiple samples. In one patient, the initial biopsy showed viral genotype 1, although subsequent biopsies showed complex genetic patterns, including a biopsy consistent with viral genotype IV. CONCLUSIONS: Many viral strains associated with BKV-ISN are difficult to classify and possibly distinct from those described in kidney transplant recipients without BKV-ISN. VP-1 sequences undergo continual modification as patients are followed in time. This genetic instability could conceivably have implications for evasion of host immunity and development of resistance to antiviral drugs.

Successful retransplantation after renal allograft loss to polyoma virus interstitial nephritis.

BACKGROUND: Although polyoma virus infection is being increasingly recognized as a cause of renal allograft dysfunction and failure, the risk of polyoma recurrence in a subsequent transplant is unknown. We present the first reported case of successful retransplantation after polyoma virus-induced renal allograft loss. CASE REPORT: A 40-year-old Caucasian woman received a cadaveric kidney transplant. Baseline immunosuppression included corticosteroids, mycophenolate mofetil, and tacrolimus. Her post-transplant clinical course was complicated by an early acute rejection episode on posttransplant day (PTD) 6, that warranted treatment with OKT3. A biopsy performed on PTD 154 to evaluate a rise in creatinine revealed polyoma virus interstitial nephritis. Despite reduction in immunosuppression, the renal function progressively worsened and dialysis was initiated by PTD 160, followed by transplant nephrectomy on PTD 184. Four months later, she received a living related kidney from her sister. Immunosuppression was initiated with prednisone, azathioprine, and tacrolimus. She had immediate graft function with a decrease in serum creatinine from 12.8 to 1.1 mg/dl. Three and one-half years after her second renal transplant, her allograft functions well, with a serum creatinine of 1 mg/dl. Both quantitative and qualitative assays of blood and urine (by PCR) remain negative for BK virus, indicating the absence of virus reactivation. CONCLUSION: Judicious retransplantation should be considered as a therapeutic option in the management of polyoma virus induced graft failure. Previous graft loss secondary to polyoma virus infection is not a contraindication to retransplantation.

Quantitative viral load monitoring and cidofovir therapy for the management of BK virus-associated nephropathy in children and adults.

BACKGROUND: BK virus (BKV)-associated nephropathy (BKVAN) has been increasingly recognized as an important cause of renal transplant dysfunction. We report the role of quantitative viral load monitoring in the management of BKVAN. METHODS: We developed a real-time quantitative polymerase chain reaction (PCR) assay for BKV detection in urine and plasma. Four renal allograft recipients, including two children, with BKVAN were treated with low-dose cidofovir and followed prospectively. RESULTS: The PCR assay showed a detection limit of 10 viral copies with an intra-assay coefficient of variation of 19%. All four patients with BKVAN demonstrated intranuclear inclusions on allograft biopsy and a progressive rise in serum creatinine; three patients underwent multiple biopsies before the diagnosis of BKVAN was made. Three of the patients experienced a "viral syndrome" before the onset of renal dysfunction. One child also demonstrated an echogenic renal mass. All of the patients demonstrated strongly positive urinary PCR values (>100,000 copies/microL). BKV DNA was also detected in the plasma of three patients. All the patients were treated with intravenous low-dose cidofovir (0.25-1 mg/kg per dose, every 2-3 weeks, without probenecid). BK viruria resolved within 4 to 12 weeks (after 1-4 doses) of the cidofovir therapy, and all patients remain with stable renal function 6 to 26 months posttherapy. CONCLUSIONS: Quantitative PCR for BKV is a sensitive and reliable method for following the course of the infection in renal transplant patients. In addition, cidofovir therapy may be useful in the treatment of some of these patients, and its role needs to be investigated further.

Antihypertensive and renoprotective efficacy and safety of losartan. A long-term study in children with renal disorders.

BACKGROUND: Hypertension is a major risk factor for progressive renal failure. We assessed the long term efficacy and safety of losartan in lowering blood pressure (BP) and in preserving renal function in hypertensive children with chronic renal disorders. METHODS: Losartan was used in 45 consecutive hypertensive children with chronic renal parenchymal disorders and mean glomerular filtration rate (GFR) 99.4 mL/min/1.73 m(2). Of the children, 21 had hypertension alone (H) and 24 had both hypertension and proteinuria (H+P). Assessment was done at baseline and at preselected time points: visit I, <0.25 years; visit II, >/=0.25 and <0.5 year;visit III, 0.5 to 1.0 year; and visit IV >1 year. Both BP control and GFR were the principal outcome measures, and proteinuria was a secondary outcome measure. RESULTS: The mean age was 12.85 years and follow-up was 2.42 years (visit IV). Compared with baseline the systolic, diastolic, and mean arterial BP (MABP) fell by 9 to 12 mm Hg (all P < .01) in visit I. Diastolic BP and MABP remained significantly lower in all visits (P < .05 to .001), whereas systolic BP was not statistically lower in visit II. In visit IV the proportion of normotensive children increased significantly compared with baseline (P < .03 for systolic BP, P < .0004 for diastolic BP). In the H+P subgroup, optimal reduction in proteinuria ranging from 66% to 71% occurred in visits II to IV (all P < .01). Mean GFR declined at a rate of 9.3 mL/min/1.73 m(2) /year before starting losartan, and 1.4 mL/min/1.73 m(2) /year subsequently (P = NS). On long term follow-up, GFR fell by 15.9 mL/min/1.73 m(2) in the H subgroup and by 5.5 mL/min/1.73 m(2) in the H+P subgroup (P = NS). There was no correlation between BP measures and GFR or between the magnitude of BP lowering and proteinuria. Adverse effects (one serious) led to discontinuation of losartan in five children (11%). CONCLUSIONS: Losartan therapy was associated with prolonged and sustained antihypertensive and renoprotective benefits in children with a variety of chronic renal parenchymal disorders. Such benefit may be more pronounced in children with combined hypertension and proteinuria. The agent was well tolerated in the majority of the children.

Evaluation of the specificity and sensitivity of a potential rapid influenza screening system.

Influenza remains a serious worldwide health threat with numerous deaths attributed to influenza-related complications. It is likely that transmission of influenza and both the morbidity and mortality of influenza could be reduced if inexpensive but reliable influenza screening assays were more available to the general public or local medical treatment facilities. This report provides the initial evaluation of a pilot system designed by Lucigen Corp. (Middleton, WI, USA) as a potential rapid near point-of-care screening system for influenza A and influenza B. The evaluation of specificity and sensitivity was conducted on stored nasal swab samples collected from emergency department patients presenting with influenza-like symptoms at a large military academic hospital and on de-identified nasal swabs and isolated RNA from a local epidemiology laboratory. The gold standard for assessment of specificity and sensitivity was the Luminex® Respiratory Viral Panel.

Pediatric living donor kidney transplantation under alemtuzumab pretreatment and tacrolimus monotherapy: 4-year experience.

BACKGROUND: Alemtuzumab has been used in off-label studies of solid organ transplantation. METHODS: We analyzed the first 42 pediatric consecutive living donor kidney transplantations under alemtuzumab pretreatment with tacrolimus monotherapy and subsequent spaced weaning. We focused especially on the causes of recipient death and graft loss and the characteristics of rejection. RESULTS: Laparoscopic live-donor nephrectomy was associated with no mortality and no delayed graft function. The actuarial 1, 2, 3, and 4 years patient and graft survivals were 97.6% and 97.6%, 93.5% and 85.4%, 93.5% and 85.4%, and 93.5% and 85.4%, respectively. The incidence of cumulative acute cellular rejection (ACR) at 1, 2, 3, and 4 years was 0%, 2.4%, 4.8%, and 4.8%, respectively. The mean serum creatinine (mg/dL) and glomerular filtration rate (mL/min/1.73 m) at 1, 2, and 3 years were 0.8+/-0.4 and 94.0+/-36.8, 0.9+/-0.4 and 79.6+/-31.9, and 0.9+/-0.4 and 95.0+/-21.7, respectively. Two (4.7%) recipients had ACR, and both Banff 1a ACRs were steroid sensitive. No patients had antibody-mediated rejection. Weaning to spaced dose (qod or less) tacrolimus monotherapy was attempted in 16 (38%) and was successful in 12 (26%) patients. All patients are currently steroid free. There was no tissue invasive cytomegalovirus disease or infection, no BK/polyoma viral nephropathy, and no posttransplant proliferative disease. CONCLUSION: This experience confirms the 4-year safety and efficacy of this approach in pediatric recipients.

Polyomavirus BK non-coding control region rearrangements in health and disease.

BACKGROUND: BK virus is an increasingly recognized pathogen in transplanted patients. DNA sequencing of this virus shows considerable genomic variability. METHODS: To understand the clinical significance of rearrangements in the non-coding control region (NCCR) of BK virus (BKV), we report a meta-analysis of 507 sequences, including 40 sequences generated in our own laboratory, for associations between rearrangements and disease, tissue tropism, geographic origin, and viral genotype. RESULTS: NCCR rearrangements were less frequent in (a) asymptomatic BKV viruria compared to patients viral nephropathy (1.7% vs. 22.5%), and (b) viral genotype 1 compared to other genotypes (2.4% vs. 11.2%). Rearrangements were commoner in malignancy (78.6%), and Norwegians (45.7%), and less common in East Indians (0%), and Japanese (4.3%). A surprising number of rearranged sequences were reported from mononuclear cells of healthy subjects, whereas most plasma sequences were archetypal. This difference could not be related to potential recombinase activity in lymphocytes, as consensus recombination signal sequences could not be found in the NCCR region. CONCLUSIONS: NCCR rearrangements are neither required nor a sufficient condition to produce clinical disease. BKV nephropathy and hemorrhagic cystitis are not associated with any unique NCCR configuration or nucleotide sequence.