A novel biomarker of laminin turnover is associated with disease progression and mortality in chronic kidney disease.

BACKGROUND: Patients with chronic kidney disease (CKD) have increased risk of development of end-stage renal disease (ESRD) and early mortality. Fibrosis is the central pathogenic process in CKD and is caused by dysregulated extracellular matrix (ECM) remodeling. The laminin γ1 chain (LAMC1) is a core structural protein present in the basement membrane of several organs, including the kidneys. We hypothesized that dysregulation of LAMC1 remodeling could be associated with a higher risk of adverse clinical outcomes in patients with CKD. METHODS: A novel immunoassay targeting LG1M, a specific MMP-9-generated neo-epitope fragment of LAMC1, was developed and used to measure the levels of the fragment in urine and serum from 492 patients from the Renal Impairment in Secondary Care (RIISC) study, a prospective cohort of patients with high-risk CKD. Patients were monitored for a median follow-up time of 3.5 years. Associations between serum and urine LG1M levels and progression of CKD at 12 months were assessed by a multivariable logistic regression model. The association with ESRD or mortality was assessed by Kaplan-Meier survival curves and Cox proportional hazards regression. RESULTS: Forty-six (11%) of the 416 patients who reached 12-month follow-up had progression of CKD; during the study follow-up, 125 patients (25.4%) developed ESRD and 71 patients (14.4%) died. Serum and urine levels of LG1M correlated with baseline eGFR (r = -0.43, p<0.0001 and r = -0.17, p = 0.0002, respectively). Serum levels of LG1M were higher in patients with one-year progression of CKD compared to those who did not progress (p<0.01). Baseline serum levels of LG1M were associated with development of ESRD (HR 3.2, 95% CI 1.99-5.2 for patients in the highest LG1M tertile compared to patient in the lowest tertile). Baseline urinary levels of LG1M (uLG1M) were significantly associated with mortality (HR 5.0, 95% CI 2.8-8.9, p<0.0001 for patients in the highest LG1M tertile compared to patients in the lowest tertile). Urine LG1M was retained in the model for prediction of mortality (HR per standard deviation of uLG1M: 1.01, 95% CI 1.00-1.02, p = 0.001). CONCLUSIONS: LG1M, a marker of basement membrane remodeling, is increased in serum and urine of patients with CKD and levels are associated with one-year disease progression, development of ESRD, and mortality.

Prostate cancer biomarker annexin A3 detected in urines obtained following digital rectal examination presents antigenic variability.

OBJECTIVES: Annexin A3 (ANXA3) is a potential marker for prostate cancer (PCa). We aimed to develop robust immunoassays suitable for quantifying ANXA3 in urine samples obtained following digital rectal examination (DRE) in order to facilitate the diagnostic performance evaluation of this marker. DESIGN AND METHODS: Anti-ANXA3 monoclonal antibodies were generated and their epitopes mapped. Two different ANXA3 assay prototypes were established on the VIDAS® automated immunoanalyser and analytical validation was carried out using post-DRE urine samples obtained from patients with PCa (n=23) or benign prostate hyperplasia (n=31). RESULTS: The assays had the same capture antibody (TGC44) but different detection antibodies (13A12 or 5C5), recognizing novel distinct epitopes. Both had a lower limit of quantification <1ng/mL and were highly specific for ANXA3, not cross-reacting with other annexins. Interassay imprecision was ≤11% and ≤15% for 13A12 and 5C5 assays, respectively. Surprisingly, a total lack of correlation was observed between ANXA3 levels measured by these two assays in post-DRE urines, indicating detection of distinct antigenic variants. Two freeze-thaw cycles did not affect analyte stability in either assay, whereas a lack of stability of antigenic variants was observed when samples were stored at -80°C for 1month. CONCLUSIONS: Two different antigenic variants of ANXA3 are present in post-DRE urines and their clinical significance for diagnosis of prostate cancer should be further investigated. These variants are not stable over time in samples preserved at -80°C. Until this issue is resolved, ANXA3 should only be measured in freshly collected samples.

Measurement of matrix metalloproteinase 9-mediated collagen type III degradation fragment as a marker of skin fibrosis.

BACKGROUND: The current study utilized a Bleomycin-induced model of skin fibrosis to investigate the neo-epitope CO3-610 (KNGETGPQGP), a fragment of collagen III released during matrix metalloproteinase-9 (MMP9) degradation of the protein, we have previously described as a novel biomarker for liver fibrosis. The aim was to investigate CO3-610 levels in another well characterised model of fibrosis, to better describe the biomarker in relation to additional fibrotic pathologies. METHODS: Skin fibrosis was induced by daily injections of Bleomycin to a total of 52 female C3 H mice, while control mice (n = 28) were treated with phosphate buffered saline (PBS), for 2, 4, 6 or 8 weeks. Skin fibrosis was evaluated using Visiopharm software on Sirius-red stained skin sections. Urine ELISA assays and creatinine corrections were performed to measure CO3-610 levels. RESULTS: CO3-610 levels were significantly higher in Bleomycin-treated vs. PBS-treated mice at each time point of termination. The mean increases were: 59.2%, P < 0.0008, at 2 weeks; 113.5%, P < 0.001, at 4 weeks; 136.8%, P < 0.0001 at 6 weeks; 157.2%, P < 0.0001 at 8 weeks). PBS-treated mice showed a non-significant increase in CO3-610 levels (mean increase for weeks 2-8 = 1.7%, P = 0.789) CO3-610 levels assayed in urine were statistically significantly correlated with Western blot analysis showing increased skin fibrosis (P < 0.0001, r = 0.65). CONCLUSION: Increased levels in mouse urine of the MMP-9 mediated collagen III degradation fragment CO3-610 were correlated with skin fibrosis progression, suggesting that CO3-610 may be a potential positive biomarker to study the pathogenesis of skin fibrosis in mice.

Association between concentrations of urinary type II collagen neoepitope (uTIINE) and joint space narrowing in patients with knee osteoarthritis.

OBJECTIVE: To examine whether urine concentrations of type II collagen neoepitope (uTIINE) distinguish subjects with progressive radiographic and/or symptomatic knee osteoarthritis (OA) from those with stable disease. METHODS: Subjects were 120 obese middle-aged women with unilateral knee OA who participated in a 30-month randomized-controlled trial of structure modification with doxycycline, in which a standardized semiflexed anteroposterior view of the knee was obtained at baseline, 16 months and 30 months. Subjects were selected from a larger sample to permit a priori comparisons between 60 OA progressors and 60 nonprogressors, as defined by joint space narrowing (JSN) in the medial tibiofemoral compartment. Each group contained 30 subjects who exhibited clinically significant increases in knee pain over 30 months and 30 who did not. Urine samples were obtained every 6 months for determination of the creatinine (Cr)-adjusted uTIINE concentration. RESULTS: Baseline uTIINE levels were unrelated to JSN in the placebo group. However, among subjects in the active treatment group, a 1-standard deviation increment in baseline uTIINE (68 ng/mM Cr) was associated with a marginally significant, two-fold increase in the odds of progression of JSN (odds ratio 2.04, 95% confidence interval 0.98-4.28). The within-subject mean of uTIINE values at baseline, 6 months and 12 months was associated with concurrent JSN measured at 16 months (0.10mm of JSN per 69 ng/mM Cr, P=0.008). Similar results were seen in the interval between months 16 and 30 and in analyses using the maximum of intercurrent uTIINE levels. CONCLUSION: Baseline uTIINE was not a consistent predictor of JSN in subjects with knee OA. However, serial measurements of uTIINE reflect concurrent JSN.

The assessment of cartilage degradation in vivo: development of an immunoassay for the measurement in body fluids of type II collagen cleaved by collagenases.

A monoclonal antibody has been developed which recognizes a neoepitope in type II collagen which is generated by the intrahelical cleavage of collagenases. Antibody reactivity is directed at the carboxyl-terminus of the TCA or 3/4 piece of the degraded alpha1(II) chain. Reactivity is dependent upon hydroxylation of proline. Evidence is provided suggesting that epitope binding involves the recognition of a conformational neoepitope. Using an ELISA, we show that this neoepitope can be detected in the urines and sera of nonarthritic persons and patients with rheumatoid arthritis (RA). An increased content is observed in the sera and urines of patients. The assay may be of value in studying cartilage type II degradation both in vitro and in vivo such as in those with arthritis.

Urinary type II collagen neoepitope as an outcome measure for relapsing polychondritis.

Herein we describe the case of a man who was diagnosed as having relapsing polychondritis (RP) when he was 18 years of age and was treated over the course of 2 years with numerous immunosuppressive agents, including tumor necrosis factor alpha (TNFalpha) inhibitors. His respiratory symptoms were refractory to treatment. Serum and urine samples were obtained periodically for measurement of erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels, anti-type II collagen (anti-CII) antibodies, and urinary type II collagen neoepitope (uTIINE) levels. The uTIINE assay is specific for collagenase cleavage products CII present in urine. ESRs and CRP levels varied widely but were rarely normal. Anti-CII antibody titers were high initially and decreased slowly and steadily for a year following the start of immunosuppressive medication, remaining low throughout the remainder of the patient's monitored disease course. The uTIINE levels were elevated prior to the initiation of TNFalpha inhibitors. Upon initiation of etanercept, they decreased abruptly to normal and stayed nearly normal. The uTIINE levels rose abruptly again upon discontinuation of TNFalpha inhibitor treatment. The dramatic decline in CII degradation, coincident with the administration of the TNFalpha inhibitors, suggested that this treatment dramatically reduced the chondritis. Serum levels of Th1 cytokines (interferon-gamma, interleukin-12 [IL-12], and IL-2) paralleled changes in uTIINE levels, while those of Th2 cytokines (IL-4, IL-5, IL-6, and IL-10) showed little or no association with disease state or uTIINE levels. These results indicate that RP might be a Th1-mediated disease process. Moreover, the uTIINE assay appears to provide an objective measure of the severity of chondritis that could assist clinical decisions regarding adjustments of steroid and other immunosuppressive therapy. This outcome measure merits investigation in a broader spectrum of RP patients.

An analysis of 14 molecular markers for monitoring osteoarthritis: segregation of the markers into clusters and distinguishing osteoarthritis at baseline.

OBJECTIVE: To investigate the relationships between serum and urinary molecular markers (MM) used to monitor osteoarthritis. DESIGN: Forty osteoarthritis patients had blood and urine collected at baseline and 1, 3, 6 and 12 months later. Specimens from 20 controls were obtained twice at a one month interval. The concentration of 14 different markers was determined at each time point and the data were analyzed by statistical methodology. RESULTS: The markers could be divided by the method of principal components analysis into five clusters of related markers: inflammation markers (C-reactive protein, tumor necrosis receptor type I and tumor necrosis receptor type II, interleukin 6, eosinophilic cationic protein), bone markers (bone sialoprotein, hydroxylysyl pyridinoline, lysyl pyridinoline), putative markers of cartilage anabolism (carboxypropeptide of type II procollagen, hyaluronan, epitope 846) and catabolism (keratan sulfate, cartilage oligomeric matrix protein), and transforming growth factor beta. Three markers (tumor necrosis factor receptor II, cartilage oligomeric matrix protein and epitope 846) from independent clusters discriminated osteoarthritis patients from controls. Inflammation was not a confounding factor in measurement, but a recognizable distinguishing factor in osteoarthritis. CONCLUSIONS: The markers separated into rational groups on the basis of their covariance, a finding with independent biochemical support. The covariance of markers from the same cluster suggests the use of a representative marker from the cluster to reflect changes in osteoarthritis. If multiple markers are being measured within a single cluster, then the use of a weighted cluster 'factor' may be preferable to the separate use of individual markers.

Measuring human chorionic gonadotropin in the absence of implantation with use of highly sensitive urinary assays for intact beta-core and free beta epitopes.

OBJECTIVE: To determine if human chorionic gonadotropin (hCG) can be absorbed from the uterine cavity in the absence of an embryo. DESIGN: Prospective study. SETTING: University-based assisted reproduction program. PATIENT(S): Eight functionally agonadal patients (age range, 33-46 years) who were taking hormone replacement therapy so that they could receive donated oocytes. INTERVENTION(S): Intrauterine instillation of 50 microL of hCG (10,000 IU) during a mock cycle before an attempt at oocyte donation. MAIN OUTCOME MEASURE(S): Spot urine measurements of different hCG epitopes (intact beta, beta-core, and free beta) at timed intervals (12, 20, 44, and 68 hours after instillation). RESULT(S): All hCG epitopes were detected in the urine at the first sampling interval, and levels decreased in subsequent sampling intervals. Measurement of the serum hCG level confirmed that systemic absorption had occurred and that the urine measurements were not a result of specimen contamination through the cervix. CONCLUSION(S): hCG may be systemically absorbed into the blood through the uterine cavity, even in the absence of implantation, and its metabolites may be measured with use of highly sensitive urinary assays.

Short report: detection of 72-75-kD and 123-kD fractions of Leishmania antigen in urine of patients with visceral leishmaniasis.

Two polypeptide fractions of 72-75 kD were detected in the urine of 14 of 15 patients with visceral leishmaniasis (VL) and another fraction of 123 kD was found in 10 of the 15 patients by using a Western blot technique. None of these fractions was detected in the urine of 20 controls. These results suggest that antigen detection in urine could be a powerful, noninvasive method for VL diagnosis.

Common epitope on urinary antigen derived from different Legionella pneumophila serogroup 1 strains recognized by a monoclonal antibody.

Guinea pigs were infected intraperitoneally with 4 subgroup reference strains (Knoxville 1, Philadelphia 1, Bellingham 1, OLDA) and 2 clinical isolates of Legionella pneumophila serogroup 1. Antigenuria was demonstrated by the enzyme-linked immunosorbent assay using polyclonal antibodies and monoclonal antibody (mab) F8/5. Mab F8/5 recognizes a hitherto undetected common epitope on urinary antigen of the investigated strains.

Conformational correlates of the epitopes of human myelin basic protein peptide 80-89.

Different epitopes residing within the decapeptide of residues 80-89 of human myelin basic protein (MBP) exist in the MBP-like material detected in human CSF and urine. In the present study, the structure of human MBP peptide 80-89 was examined by a combination of physical measurements and correlated with its varying immunochemical reaction with three polyclonal antisera. At least two epitopes are present in the decapeptide. Progressive shortening and reduction in net negative charge of MBP peptide 80-89 to form peptides 81-89, 82-89, 83-89, and 84-89 revealed an epitope not present in intact MBP. Circular dichroism and Fourier-transform infrared of these MBP peptides in water demonstrated random structure that was partially changed to beta-structure in the shorter peptides. In methanol, used as a model for a lipid environment, the random structure was diminished and was replaced by alpha-helix and beta-structure, especially in the shorter peptides. The findings indicate that the range of epitopes present in this decapeptide is influenced by conformation, which, unexpectedly, becomes progressively less random as the peptide becomes smaller, especially in a hydrophobic environment. This behavior has implications for the immunochemical detection of small antigens or antibodies to them in tissue extracts or body fluids.