It is well known that change in apoptosis may modulate the natural story of illness, and that many drugs may act through modulation of apoptosis, but the role of steroids in acting through apoptosis in different settings, including renal diseases, has still to be elucidated. We studied the in vivo effects of steroids by oral assumption (10 to 25 mg/deltacortene) or by intravenous pulses (300 to 1000 mg/dose) on apoptosis and cellular subsets of peripheral lymphocytes, by evaluating DNA-fragmentation and lymphocyte subsets in 79 subjects: 22 controls and 57 patients with various renal diseases (25 Lupus-GN, 19 membranous-GN (MGN), 6 rapidly progressive-GN (RPGN), 2 acute interstitial nephritis (AIN), 5 on chronic dialysis. Baseline apoptosis was present in 1/22 (4.5%) of controls, 3/25 (12%) SLE, 2/6 (33.3%) RPGN and 10/19 (52.6%) MGN. A significant decrease in CD3+CD8+ cell count and a significant increase of the CD3+CD4/CD3+CD8+ ratio were found in apoptosis-positive subjects. DNA fragmentation did not change after oral steroids, paralleling a 22 to 32% decrease in total lymphocytes. Following intravenous methylprednisolone pulses, a deeper drop of all lymphocyte subsets was observed, while DNA fragmentation turned from present to absent in 2 MGN, but not in 2 RPGN, and from absent to present in 1 ARF and 1 SLE, independently of the dosage. We demonstrated that the presence of apoptosis in renal diseases is associated with decreased CD3+CD8+ cell count. Furthermore, steroid intravenous pulses, besides inducing a profound decrease in lymphocyte subsets, do exert a dual effect on baseline leukocyte apoptosis, eventually leading to a reversal of baseline patterns, either turning from negative to positive or from positive to negative. Oral steroid therapy did not influence baseline apoptosis.

rum samples and different calibration slopes for human plasma and mouse/rat plasma.They observed an overestimation of ϳ20% for serum ADMA concentrations determined by the ELISA compared with LC-MS.Our results indicate a higher discrepancy between the 2 methods, possibly because our comparison used plasma samples, which have a different matrix effect on the ELISA results.The matrix dependence of the ELISA may be the cause of the calibration offsets.The calibration must be performed with the supplied calibration samples, which consist of ADMA in buffer solution.The systematically observed errors in the calibration may arise because these calibration samples are very different from plasma samples.Overall, the ELISA appears to overestimate ADMA concentrations in plasma by a factor of nearly 2. In the group of healthy individuals, the mean (SD) ADMA concentration determined with the ELISA was 0.720 (0.111) mol/L.Schulze et al. (10 ) found serum ADMA concentrations of 0.72 (0.16) mol/L in healthy volunteers.The corresponding values for comparable groups described in recent studies focusing on analytical technology (8, 11-13 ) were 0.355, 0.42, 0.39, and 0.343 mol/L.The precision of the ELISA was good.We obtained a relative SD of 6.1% when we measured a sample from the healthy group 10 times.This is comparable to the value of 7.5% reported by Schulze et al. (10 ) and slightly above the value of 4.7% reported for the LC-MS assay (8 ).The workloads for the laboratory personnel are comparable for the ELISA and the LC-MS assay.In the case of the ELISA, 20 L of each sample must be pipetted into the microtiter plate well and acylated for 1.25 h.The ELISA process then takes place overnight in a refrigerator.The actual measurement of samples takes only seconds.Processing of a complete 12 ϫ 8 microtiter plate begun in the afternoon is completed the next morning.The sample preparation for the LC-MS assay consists of pipetting the samples, addition of the internal standard, protein precipitation, centrifugation, evaporation of the samples under reduced pressure, and redissolution of the residue.The overall time required for preparing 40 samples is ϳ3 h, with ϳ1.5 h of hands-on time.The subsequent LC-MS determination time is 24 h for 40 samples with the system described by .In conclusion, the ADMA ELISA is suitable for clinical investigations in which groups of samples are compared and the endpoint is the shift of the ADMA concentration in response to an intervention.Differentiation between sample groups measured with the ELISA is less reliable than with LC-MS because the CV is higher and the matrix dependence can cloud or mimic differences.Overestimation of ADMA concentrations also makes comparison with data in the literature very difficult.Expenses for consumable items are clearly higher for the ELISA than for the LC-MS assay, a situation that is aggravated by the necessity of measuring Arg (3 ) separately because this important analyte is not measured by the ELISA, whereas in the LC-MS assay it can be measured in the same run as ADMA with no additional costs.On the other hand, higher sample throughput is possible with the ELISA.

To the Editor: After reading the paper by Wrone et al in a recent issue of Kidney International regarding methylenetetrahydrofolate reductase (MTHFR) variant and cardiovascular disease (CVD) in renal patients1 we have some concerns we would like to express.

Anti-endomysium antibody (EmA) testing is used in the diagnosis of celiac disease (CD). The identification (1) of tissue transglutaminase (tTG) as the main antigen of EmA led to the development of commercial ELISAs for serum anti-tTG detection. At first, a guinea pig antigen (2)(3)(4)(5)(6)(7) yielded both sensitivity and specificity lower than those of EmA; therefore, human recombinant tTG (8)(9)(10)(11) was introduced to improve diagnostic accuracy and to overcome problems such as species specificity and cross-reactivity to contaminant proteins. However, the standardization of assays (12), the choice of cutoff value, the clinical relevance of these autoantibodies (13)(14), and the diagnostic accuracy of different commercial tests remain unresolved (15)(16)(17)(18). This study aimed to assess the diagnostic accuracy of five commercially available IgA anti-tTG ELISA reagent sets (four using a human recombinant and one a guinea-pig tTG antigen) for pathologically confirmed CD and to evaluate the changes in anti-tTG autoantibody concentrations during treatment of CD with a gluten-free diet (GFD). This prospective study included 101 consecutive untreated adults (79 women and 22 men; median age, 37 years; range, 21–72 years) referred to University gastroenterologic outpatient clinic between January 2000 and May 2001 in whom CD was subsequently diagnosed by means of the typical appearance of small intestinal mucosa (19) (Marsh grade III in 95 patients and grade II in 6 patients) and by a positive clinical response to a GFD. We reported on 34 of these patients (all EmA-positive) previously (17). A duodenal biopsy was performed in all patients on the basis of clinical history and serologic assessment, including EmA testing and nutritional indexes. In all patients, a follow-up biopsy and serologic monitoring were repeated at 1 year ± …

BACKGROUND:Parathyroid hormone (PTH) has important applications in the nephrological clinical practice. Because assays of Intact PTH (I-PTH) are liable to interferences by N-truncated fragments, a novel method for whole-(1-84) PTH has been proposed. This study is aimed at comparing the latter with some of the previous I-PTH assays. For each method the results are referred to pertinent markers of mineral metabolism.METHODS:We enrolled 171 subjects, including 56 healthy controls (C), 65 calcium stone- formers (CaSF), 40 haemodialysis patients (HD), 10 with primary hyperparathyroidism (PHP). On blood samples we measured: I-PTH by four methods (N-Tact, Advantage, Elecsys, Scantibodies), whole-(1-84) PTH, defined as CAP (Cyclase Activating PTH), total and ionised calcium, phosphate, vitamin D, osteocalcin and Crosslaps. The difference between I-PTH and CAP Scantibodies is defined as CIP (Cyclase Inhibiting PTH).RESULTS:Despite relating to each other (r>0.97) PTH values varied remarkably among methods. For all methods, the reference intervals differed from those provided by the producer. Assuming these new ranges, 10 CaSF had over-range values not always associated with abnormalities of mineral metabolism. One of the PHP patients was normal for I-PTH with 2/4 methods. In HD the differences among methods were even greater, there were inverse (p<0.05) and direct (p<0.001) relationships with ionised calcium and osteocalcin-crosslaps, respectively. The CAP/CIP ratio was lower in low bone turnover patients, but the two subgroups widely overlapped.CONCLUSIONS:This study indicates that the reliability of I-PTH assays is still unsatisfactory, and none of the four methods emerged as the best. Assay for CAP only improves diagnostic efficiency, whereas the CAP/CIP ratio does not exhibit powerful discriminating capacity. Our suggestion is that each Centre should establish its own reference ranges. PTH assay should always be coupled with measurements of other markers of mineral metabolism as well as renal function.

BACKGROUND AND AIMS Duration of gluten exposure seems to predispose adolescents with coeliac disease to autoimmune diseases. In a retrospective cohort study, we assessed the relationship between autoimmune disorders and actual gluten exposure in patients in whom coeliac disease was diagnosed in adult life (⩾16 years). METHODS We screened for the presence of autoimmunity in 605 controls (16–84 years) and 422 patients (16–84 years), all of whom had been on gluten withdrawal for at least one year (median follow up 9.5 years). A logistic regression analysis, setting the prevalence of autoimmunity as the dependent variable, was employed to control for independent covariates as predictors of the risk of autoimmunity. RESULTS The prevalence of autoimmunity was threefold higher (p<0.00001) in patients than in controls. Mean duration of gluten exposure was 31.2 and 32.6 years for patients with or without autoimmunity. Logistic regression showed that increased age at diagnosis of coeliac disease was related to the prevalence of autoimmune disease while "actual gluten exposure" which takes into account diet compliance, follow up, and age at diagnosis of autoimmune disorders were not predictive for the risk of developing autoimmune diseases (odds ratio 0.82 per year). CONCLUSION The prevalence of autoimmune diseases in patients with a late coeliac disease diagnosis does not correlate with duration of gluten intake. Early exposure to gluten may modify the immunological response. Gluten withdrawal does not protect patients with a late diagnosis from autoimmune diseases.

Background: The study was aimed at comparing the diagnostic accuracy of the quantitative bladder tumor antigen (BTA) TRAK™ immunoassay with exfoliative urine cytology in the detection of primary and recurrent bladder cancer. Methods: The analysis was carried out on 194 high risk patients undergoing a diagnostic cystoscopy, 279 patients with previous history of transitional cell carcinoma awaiting a follow-up cystoscopy, and 45 healthy controls. Urine cytology was performed by a skilled cytopathologist on three consecutive samples. Results: BTA TRAK values resulted significantly higher in tumor positive cases than in absence of bladder tumor for both groups of patients. Non neoplastic urothelial diseases as well as the absence of mucosal abnormalities were associated with a marked increase in BTA TRAK levels with respect to the control group. Overall sensitivity and specificity was 63 and 63% for BTA TRAK (cut-off 34 U/ml), and 68.3 and 73.4% for urine cytology, respectively. The diagnostic advantage of urine cytology was maintained when patients were stratified by tumor grade. Conclusions: The clinical performance of the BTA TRAK in the detection of primary or recurrent bladder cancer is acceptable and reproducible as shown by similar results with previous reports, although urine cytology performed on three samples showed the highest sensitivity and specificity.

Ͻ0.05 for all comparisons).Peak concentrations of Ntx were found during the second 2400 -0800 sampling [1323.3(134.4)nmol/mmol] and trough concentrations during the 2000 -2400 time period [812.1 (98.8) nmol/ mmol; P Ͻ0.01].Ntx in both samples collected at 2400 -0800 were higher than in the samples collected at 1200 -1600, 1600 -2000, and 2000 -2400 (P Ͻ0.05 for all comparisons).Considering the marked diurnal variations in PICP and ICTP, it may be difficult to interpret the finding of no rhymicity in PINP concentrations.There are no available data in adults or in children for comparison of the finding.Perhaps differences in assay sensitivity and metabolism of the propeptides may play a role, necessitating a larger study population than the present to detect possible variations in PINP.Our observation of no diurnal variation in PIIINP concentrations, however, is in accord with a previous report in children (1 ).Using an immunoassay, a study of a group of healthy girls, 10 -14 years of age, revealed nocturnal peak concentrations of DPD at 0300 and trough concentrations at 1300 (6 ).The findings are in accord with observations of adults based on immunoassay (7 ) and HPLC (8 ) for assessment of total DPD.Furthermore, our results obtained by an immunoassay that specifically measures the free fraction of DPD are consistent with the findings of Robins et al.(4 ).One study using HPLC, however, found a statistically significant circadian variation only in total, not in free, DPD (9 ), thus supporting suggestions that in adults, diurnal variations in free DPD may be less marked than in total DPD.Whether this may reflect that the ratio between free and total DPD may vary between children and adults is unclear.Furthermore, the present finding of diurnal variations in Ntx corrected for creatinine is in accord with studies in adults (10 ).However, when Ntx was not corrected for creatinine in adults, a circadian variation could not be detected.To what extent diurnal variations in DPD and Ntx in children may depend on variations in creatinine needs further study.

Objective: Percent free prostate–specific antigen (PSA) is a promising tool for prostate cancer (CaP) diagnosis. However, its diagnostic performances have not yet been established. The present study was carried out with the aim of evaluating percent free PSA in the most favourable analytical conditions.Materials and Methods: Eighty–eight patients affected by newly diagnosed, untreated, primary CaP, and 169 cases with biopsy–confirmed, untreated, benign prostatic hypertrophy (BPH) were prospectively enrolled. Abbott AxSYM total and free PSA were measured by the same technician using the same instrument and the same reagent batch.Results: Percent free PSA was more effective than total PSA in differential diagnosis between CaP and BPH in every evaluated dose range of total PSA. In cases with total PSA >4 μg/l, percent free PSA could have reduced by about 50% the rate of unnecessary biopsies with a probably still acceptable 93% cancer detection rate. The likelihood of CaP after the determination of percent free PSA was in fact higher than 50% using cut–off points which provide low sensitivity values (i.e. 58% in men aged 50–59 years).Conclusions: Percent free PSA is superior to total PSA in distinguishing primary CaP from BPH in patients with total PSA between 2 and 30 μg/l and in reducing the rate of unnecessary biopsies in men with total PSA higher than 4 μg/l. However, percent free PSA should be cautiously interpreted in decision making in individual patients since post–test probability is relatively low in men aged 50–70 years.

Hyperthyroidism is associated with enhanced osteoblastic and osteoclastic activity, and patients frequently have low bone mineral density and high bone turnover. The aim of this study was to examine the bone formation and resorption markers trend in 12 female patients, before and after normalization of thyroid activity. The following measurements were made at baseline and 1 and 6 months after hormone normalization induced by methimazole treatment: total alkaline phosphatase (ALP), bone alkaline phosphatase (BALP), collagen type C-terminal propeptide (PICP), osteocalcin (BGP), telopeptide (ICTP), urinary-hydroxyproline/ urinary creatinine (uOHP/uCreat), urinary calcium/urinary creatinine (uCa/uCreat) and deoxypyridinoline crosslinks (D-Pyr). Compared with controls, all of these parameters were significantly increased (ALP p=0.014; BALP p=0.0001; PICP p=0.013; BGP p=0.009; ICTP p=0.0001; uOHP/uCreat p=0.002; uCa/uCreat p=0.044; crosslinks p=0.0001). After treatment the values of ALP, BALP and PICP in hyperthyroid patients showed an initial slight increase and then a significant downwards trend (ALP p=0.008, BAP p= 0.001, PICP p=0.026). Furthermore, resorption markers showed a significant decrease (uOHP/ uCreat p<0.005 and D-Pyr p<0.008). As regards lumbar BMD patients, measurements were significantly reduced in comparison with the control group (p=0.005). Six months after serum thyroid hormones level normalization, we observed a significant increase (p=0.014 vs baseline). Both neo-formation and resorption markers are useful to assess pathological bone turnover and bone involvement in hyperthyroidism. They could also be employed to monitor the effect of antithyroid treatment on bone and to indicate if bone antiresorption therapy should be considered.