Colin L. Jones

The measurement of relative antibody affinity by ELISA using thiocyanate elution

A variation of the standard ELISA assay was used to determine the relative affinities of six murine monoclonal anti-dinitrophenol (DNP) antibodies for DNP-bovine serum albumin (DNP-BSA). The procedure involved exposing replicate wells containing antibody bound to antigen to increasing concentrations of the chaotropic thiocyanate ion. Resistance to thiocyanate elution was utilized as a measure of affinity and an index (affinity index) representing a 50% reduction in initial absorbance was used to rank the anti-DNP antibodies in order of increasing affinity. For comparison, the affinity constants of the six monoclonals were determined by equilibrium dialysis with 3H-epsilon-DNP-L-lysine as antigen. A significant correlation (P less than 0.02) was observed between the ranking orders obtained using the two methods, thereby demonstrating the applicability of the elution technique for the measurement of relative antibody affinity.

Clinical spectrum of Denys-Drash and Frasier syndrome

Abstract Denys-Drash syndrome (DDS) and Frasier syndrome (FS) are two related conditions caused by mutations of the Wilms tumor gene, WT1. Both syndromes are characterized by male pseudohermaphroditism, a progressive glomerulopathy, and the development of genitourinary tumors. DDS and FS have previously been distinguished by differences in nephropathy, with DDS patients demonstrating diffuse mesangial sclerosis (DMS) in contrast to focal and segmental glomerulosclerosis (FSGS) in FS patients. The clinicopathological features and genotype analysis of two patients with WT1 mutations are presented in this report. Genotype analysis of the first patient revealed a previously undescribed mutation in exon 8 of the WT1 gene. The second patient presented with a rapidly progressive nephropathy characterized histologically by DMS, but was found to have the genetic mutation seen in FS patients. A summary of all reported patients with the characteristic mutation associated with FS demonstrates the clinical overlap of this syndrome with DDS. This suggests that both these conditions should be considered as part of the spectrum of disease due to WT1 gene mutations rather than as separate diseases. Clinical classification remains important for prognosis, as the underlying renal disease appears to predict the progression of nephropathy independently of the genetic abnormality.

Evaluation and long-term outcome of pediatric renovascular hypertension

Abstract Seventeen children with renovascular hypertension were managed at the Royal Childrens Hospital, Melbourne, over the 20-year period from 1975 to 1996. The age at presentation ranged from 10 days to 18 years. All children presented with severe hypertension with mean systolic blood pressure 7 standard deviations above age-matched averages and mean diastolic blood pressure 5.5 standard deviations above age-matched averages. Neurofibromatosis was the most common etiology (58% of patients) and there were no cases of Takayasus arteritis. Patients underwent a variety of biochemical and imaging investigations but in all cases renal angiography was necessary for definitive diagnosis and for planning therapy. Ten of the 17 patients had surgical procedures performed. Percutaneous transluminal angioplasty was performed in four patients but led to cure in only one patient following thrombosis of the affected artery producing segmental renal infarction. Other vascular reconstructive procedures, including the use of autologous or synthetic bypass grafts and autotransplantation, produced cure of hypertension in 50% of children with improvement in a further 30%. The long-term outlook for children treated with surgical reconstructive procedures was excellent. One patient underwent surgery for avulsion of an arterial graft following a pubertal growth spurt. No other patient originally cured by surgery has required reoperation with no cases of restenosis at a mean follow-up of 11 years 3 months.

Familial hypomagnesaemia--hypercalciuria leading to end-stage renal failure.

Several disorders of hypomagnesaemia of hetary renal origin are now recognised. The cases of two sisters from a consanguineous marriage with the syndrome of renal magnesium wasting, hypercalciuria and nephrocalcinosis are presented. Pathological examination of the heterozygous parental kidneys revealed mild focal interstitial fibrosis. This condition is a previously unreported cause of end-stage renal failure in chilhhood, and this report suggests that transplantation from heterozygous parental donors can be successfully undertaken without recurrence currence of the syndrome.

Association of renal adenocarcinoma and BK virus nephropathy post transplantation.

While most BK virus infections are asymptomatic, immunosuppression has been associated with BK virus reactivation and impaired graft function or ureteric ulceration in renal transplant patients and hemorrhagic cystitis in bone marrow transplant patients. Oncogenicity is also postulated and this is the first report of a child with a carcinoma of the donor renal pelvis following BK virus allograft nephropathy. Removal of the primary tumor and cessation of immunosuppression led to regression of secondary tumors and a return to health.

Triple immunosuppression with subsequent prednisolone withdrawal: 6 years' experience in paediatric renal allograft recipients

Thirty-four children (≤15 years of age) with end-stage renal failure received 39 renal allografts between 1985 and 1991 and were treated with cyclosporin A (CyA), azathioprine and low-dose prednisolone (PNL). We aimed to withdraw PNL by 6 months after transplantation. Median duration of follow-up was 2 years 4 months (range 0.1 month to 6 years, 4 months). There were no deaths. Crude graft survival for living-related grafts (n=9) was 100%, although only 1 patient has been followed for >2 years. For cadaveric grafts (n=30), 1- and 5-year actuarial graft survivals were 90% and 79% respectively. At 12 months posttransplant, the median (range) glomerular filtration rate for all patients was 63 (19–109) ml/min per 1.73 m2 (n=25) and at 5 years was 48 (17–64) ml/min per 1.73 m2 (n=9). Complications observed included rejection episodes which occurred after discontinuation of PNL. Long-term (after 12 months), 28% of patients remain on PNL. Hypertension was present in more than 50% of patients. Severe CyA nephrotoxicity was not seen. Catch-up growth as determined by the change (Δ) in mean height standard deviation score (Ht-SDS) was noted at 1 year [ΔSDS/year=+0.60;P<0.001 (n=18)] and at 2 years [ΔSDS/year=+0.27;P<0.01 (n=16)] in pre-pubertal patients. The median Ht-SDS at 2 years for pre-pubertal children was −0.71 SD and growth velocity did not improve thereafter. In pubertal patients, the mean ΔSDS per year at 1 year (n=7) was +0.43 and at 2 years (n=4) was +0.17. The catch-up growth in pubertal patients did not reach statistical significance. It was concluded that the use of this immunosuppression regime was associated with an excellent patient and graft survival. Catch-up growth is especially encouraging in pre-pubertal patients. However routine discontinuation of PNL may require review.

Estimating the relative avidity of mucosal IgA for antigen

This report concerns the application of a method to estimate the relative avidity of mucosal IgA for casein and beta-lactoglobulin. The chaotropic ion, thiocyanate, was used to disrupt antigen-antibody binding in an enzyme immunoassay. The resultant proportional decrease in optical density of the enzyme immunoassay was directly related to the proportion of IgA eluted from the solid phase-bound antigen. The relative avidity was expressed as the molarity of KSCN resulting in a particular reduction in optical density. The measurement of avidity, the avidity index, was independent of the concentration of mucosal IgA. For anti-casein IgA the interassay coefficient of variation of the index was 14-18%, and the intra-assay coefficient of variation was 6%. For anti-beta-lactoglobulin IgA the interassay coefficient of variation was 10-13% and the intra-assay coefficient of variation was 6%. The method is simple and free of complicated calculations.

Factors influencing growth and final height after renal transplantation

Abstract: Growth retardation occurs commonly in children and adolescents with chronic renal insufficiency. While some children exhibit catch‐up growth following renal transplantation, for many children growth remains sub‐optimal. The aim of the current study was to review the factors influencing growth and final height following renal transplantation. Data from all children who had a renal transplant performed between 1985 and 1998 at the Royal Melbourne and Royal Childrens Hospitals, Melbourne (n = 85), were examined retrospectively. Two children who died in the first year post‐transplant and one patient lost to follow‐up within 6 months of their transplant were excluded. Children with multiple grafts had only growth following their most recent graft analyzed. The mean height standard deviation score (Ht‐SDS) at the time of transplantation was −2.11 (range: −5.05 to 0.27), improving to −1.50 (range: −3.67 to 1.27) at 7 yr post‐transplant. On univariate analysis, the dose of cyclosporin at 6 months and at 1 and 3 yr, and the graft function at 1 yr, had a significant positive correlation with the change in Ht‐SDS (ΔHt‐SDS) at each of those time‐points post‐transplant. At all time‐points there was a strong correlation between pretransplant height and subsequent growth. A sub‐group of children who were 16 yr of age or older at December 1999, and who were considered to have reached their final height, were examined to determine predictors of final height. Multiple regression analysis of clinical and laboratory parameters from the sub‐group of patients ≥ 16 yr of age showed that height at the time of transplant, age at the time of transplant, and final glomerular filtration rate, were significant independent predictors of growth (r2 = 0.82, p = 0.01). In addition, the immunosuppressive regimen at 1, 3, and 5 yr post‐transplant had a significant effect on growth. This study confirms the importance of each of these factors for post‐transplant growth.

Matrix degradation in renal disease

Summary: The histological appearance of the accumulation of glomerular and tubulointerstitial extracellular matrix (ECM) is a characteristic feature of progressive renal disease. the usual processes behind this finding are a combination of relative loss of parenchymal cells with respect to ECM, increased synthesis of ECM and decreased degradation of ECM. the physiology and pathophysiology of matrix degradation form the basis of tissue remodelling in general and have only been specifically studied in relation to renal disease recently. the two major ECM degrading enzyme systems (the matrix metalloproteinase (MMP), tissue inhibitor of the MMP (TIMP) system and the plasminogen activator/plasmin system) and the interaction between these systems and other non‐specific seem to have an important role in the processes causing matrix accumulation in the ageing kidney, focal sclerosis, diabetes, glomerulonephritis with matrix accumulation and tubulointerstitial disease. A pattern of changes in the expression of components of these enzyme‐inhibitor systems marked by increased TIMP‐1, increased plasminogen activator inhibitor‐1, decreased MMP‐1 and MMP‐3, and increased MMP‐2 and MMP‐9 characterized several models of glomerular and tubulointerstitial fibrosis that are associated, and perhaps caused by, transforming growth factor β. A thorough understanding of the processes controlling matrix degradation may not only be necessary to explain the pathogenesis of matrix accumulation but may also be important in attempting to reverse the processes.

Endothelin and Endothelin A/B Receptors Are Increased after Ischaemic Acute Renal Failure

Background/Aims: Endothelin (ET) has been implicated as an indirect mediator of injury following acute renal ischaemia (ARI). The purpose of this study was to localize and quantitate ET and ETA and ETB receptors following ARI. Methods: A model of ARI, well characterized previously, was produced by 45 min occlusion of the renal pedicle of unilaterally nephrectomized female Sprague-Dawley rats. Animals were sacrificed 1, 2, 4, 8, 16, 32 and 64 days after ischaemia (n = 6). Corresponding control groups with unilateral nephrectomy but no ischaemia were sacrificed after 0, 8 and 64 days. Immunohistochemistry for ET-1, -2 and -3 was performed. Tissue ET levels were calculated by RIA (femtomoles per kidney). Receptor ligand binding studies for ETA and ETB receptors were performed by autoradiography on frozen kidney sections and quantitated by densitometry (relative optical density per square millimetre). Results: The concentration of tissue ET increased from 24 h after ischaemia and remained significantly increased for the duration of the study, reaching a maximum at 8 days. There was a small increase in the non-ischaemic 8-day control group, but this returned to basal levels by day 64. The increase in tissue ET 8 days after ischaemia was localized by immunohistochemistry to renal medullary interstitial cells, damaged tubules at the corticomedullary junction and peritubular capillaries surrounding these damaged tubules. Increases in cortical ETA and ETB receptors were evident 24 h after ischaemia and were maximal 8 days after ischaemia, before returning to basal levels at 16 days. After a small increase 24 h after ischaemia, medullary ETA receptors decreased on day 4 before returning to basal levels on day 8 after ischaemia. Medullary ETB receptors, however, decreased on day 4 after ischaemia and remained low throughout the duration of the study. Conclusion: The previously reported amelioration of pathological changes resulting from the use of ET receptor antagonists after ARI may be related to the quantitative and qualitative changes in tissue ET and ET receptors observed in this study.