Figure 2. Western blot analysis of cystatin C in the CSF of patients suffering from sCJD. Equivalent amounts of CSF protein (10 mg) from late-stage sCJD patients and from appropriate age- matched non-sCJD patients were fractionated under non-reducing SDS- PAGE conditions and analyzed by Western blot using a polyclonal anti- human cystatin C antibody. No difference was found in CSF cystatin C levels of sCJD patients vs. controls. doi:10.1371/journal.pone.0003870.g002 plasma (See Table 2), alterations may simply reflect general proteinuria resulting from compromised renal function. We addressed this in several ways. Firstly, prospectively recruited patients with known macro- or microhaematuria, or proteinuria, and retrospectively collected urine samples with albuminuria of $150 mg/ml, were excluded from further analysis. Secondly, we obtained urine from an additional set of patients (control group C) which predominantly included post-kidney-transplant patients following diabetic nephropathy or focal segmental glomeruloscle- rosis and reflux nephropathy. Thirdly, we investigated urine from patients presenting with bladder carcinoma, and with pyelone- phritis with bacteriuria (Control group C). These groups also served to assess the specificity of the changes observed in the CJD groups relative to other non-neurological conditions. Since many factors can affect urine concentration and, consequently, concentration of test substances, we normalized cystatin C and a1-ACT concentration against urinary creatinine. In the absence of renal dysfunction, the excretion rate of creatinine is relatively constant and has been used for adjusting urine concentration/dilution effects in measurement of urinary metab- olites [28]. Normalization to creatinine therefore controls for both potential variations in urine concentration between individuals in spot-test urine samples and renal function. For these reasons we also expressed cystatin C and a1-ACT concentrations relative to both albumin and creatinine (Table 3). Urinary a1-ACT levels were evident in sCJD patients regardless of normalization to urinary albumin or creatinine. Additionally, we observed that neither age, sex or study centre sampling location were variables contributing to elevated urinary a1-ACT levels in sCJD patients (not shown). Since ELISA results can be confounded by crossreactive contaminants, we performed Western blotting analyses on selected urine samples. These confirmed dramatically elevated immunore- active bands with the electrophoretic motility of a1-ACT in sCJD patients (Fig. 4A). In contrast, we observed no statistically significant alteration in urinary cystatin C between sCJD or vCJD groups and respective controls, regardless of whether cystatin C concentration was expressed as raw, non-normalized data, or relative to either urinary albumin or creatinine (Table 3). In addition, urinary a1-ACT was also significantly higher in sCJD patients compared to control group C when expressed either relative to albumin or creatinine (Table 3, p,0.001). When sCJD patients were compared to control groups A, C, and AD patients, the sensitivity and specificity of elevated urinary a1-ACT for sCJD were, respectively, 72.4%, 87.5% (mg/mg Prion Disease Biomarker albumin) or 89.7%, 100% (mg/g creatinine) if Control group C is not included in the calculation, and 79.3%, 94.7% (mg/mg albumin) or 96.6%, 100% (mg/g creatinine) if Control group C is included in the calculation (Fig 5). In contrast to the data obtained for sCJD patients, we observed no significant alterations in urinary a1-ACT from vCJD patients referenced to control group B (Table 3, Figure 3). Longitudinal measurements of two sCJD patients presented to the NRPE (one definite and one probable sCJD) revealed a progressive increase in urinary a1-ACT relative to both albumin and creatinine throughout the clinical course of disease (Fig. 4B). This finding suggests that urinary a1-ACT excretion may be useful for monitoring disease progression, (e.g. in therapeutical trials) as a responsive surrogate biomarker. As a further indication of renal proximal tubule function, we measured urinary levels of a1-microglobulin in the sCJD and vCJD groups relative to respective control groups. The concen- tration of a1-microglobulin, normalized to creatinine or albumin, were not significantly altered in sCJD urine (Table 3), yet appeared to be higher in urine of vCJD patients than in appropriate controls (p,0.001). Normalization of values to both albumin and creatinine, and comparison to control group C, indicates that the observed increase in urinary a1-ACT concen- trations in sCJD patients is independent of renal function. We also retrospectively assessed plasma creatinine and urea levels in 19 CJD patients presented to the NRPE. This analysis did not bring forward any evidence that gross renal impairment may represent a general feature of late-stage sCJD (Table 4). Urinary a1-ACT in field cases of animal prion diseases We carried out Western blot analyses of urine from scrapie-sick sheep, cattle with confirmed Bovine Spongiform Encephalopathy (BSE) and deer with confirmed Chronic Wasting Disease (CWD), along with urine from healthy animals collected post-mortem (Fig. 6A–C). In addition, we analyzed by Western blot urine samples collected sequentially from one sheep that was pre- clinically infected with scrapie and went on to display clinical signs of disease. Scrapie was confirmed post-mortem in this sheep by histopathology and Western blot analysis of brain tissue (data not shown). Urine from the latter sheep was analyzed at 10, 9, 2 & 1 months prior to the development of terminal scrapie, together with urine from six sheep unaffected by scrapie (Fig. 6D). Urinary a1-ACT was increased 2 months before terminal disease, and remained sustained thereafter. We also observed significantly elevated levels of urinary a1-ACT in two of six BSE-affected cattle (Fig. 6B) and in one of two CWD- affected deer (Fig. 6C). One animal from the sheep study shown in Fig. 6A was culled following appearance of scrapie-like symptoms. However, post-mortem biochemical and pathological confirma- tion was inconclusive, and this particular animal was classified as ‘‘atypical’’ scrapie. It is therefore unclear whether elevated urinary a1-ACT in this animal represents a false positive, or the identification of a scrapie case hitherto unqualified by other biochemical and histological techniques. Discussion A global microarray screen of scrapie-infected mouse brains identified a limited number of genes undergoing transcriptional elevation well before the clinical onset of disease. One of these was Serpin-a3n, which encodes the serine proteinase inhibitor a - 1 antichymotrypsin (a1-ACT). Its transcription was drastically elevated not only in whole-brain homogenates of scrapie-infected mice, but also in occipital cortices of sCJD patients. These PLoS ONE | www.plosone.org 5 December 2008 | Volume 3 | Issue 12 | e3870