Urinary collagen peptides: Source of markers for bone metabolic processes in kidney transplant recipients.

INTRODUCTION: Kidney transplant recipients (KTRs) are at an increased risk of fractures. Total urinary hydroxyproline excretion served as marker for bone resorption (BR) but was replaced by ß-CrossLaps (CTX), a C-terminal collagen α-1(I) chain (COL1A1) telopeptide. We investigated the low-molecular-weight urinary proteome for peptides associated with changes in bone metabolism after kidney transplantation. METHODS: Clinical and laboratory data including serum levels of CTX in 96 KTR from two nephrology centers were correlated with signal intensities of urinary peptides identified by capillary electrophoresis mass spectrometry. RESULTS: Eighty-two urinary peptides were significantly correlated with serum CTX levels. COL1A1 was the predominant peptide source. Oral bisphosphonates were administered for decreased bone density in an independent group of 11 KTR and their effect was evaluated on the aforementioned peptides. Study of the peptides cleavage sites revealed a signature of Cathepsin K and MMP9. Seventeen of these peptides were significantly associated with bisphosphonate treatment, all showing a marked reduction in their excretion levels compared to baseline. DISCUSSION: This study provides strong evidence for the presence of collagen peptides in the urine of KTR that are associated with BR and that are sensitive to bisphosphonate treatment. Their assessment might become a valuable tool to monitor bone status in KTR.

Increased Collagen Turnover Is a Feature of Fibromuscular Dysplasia and Associated With Hypertrophic Radial Remodeling: A Pilot, Urine Proteomic Study.

Fibromuscular dysplasia (FMD), a nonatherosclerotic, noninflammatory disease of medium-sized arteries, is an underdiagnosed disease. We investigated the urinary proteome and developed a classifier for discrimination of FMD from healthy controls and other diseases. We further hypothesized that urinary proteomics biomarkers may be associated with alterations in medium-sized, but not large artery geometry and mechanics. The study included 33 patients with mostly multifocal, renal FMD who underwent in depth arterial exploration using ultra-high frequency ultrasound. The cohort was separated in a training set of 23 patients with FMD from Belgium and an independent test set of 10 patients with FMD from Italy. For each set, controls matched 2:1 were selected from the Human Urinary Proteome Database. The specificity of the classifier was tested in 700 additional controls from general population studies, patients with chronic kidney disease (n=66) and coronary artery disease (n=31). Three hundred thirty-five urinary peptides, mostly related to collagen turnover, were identified in the training cohort and combined into a classifier. When applying in the test cohort, the area under the receiver operating characteristic curve was 1.00, 100% specificity at 100% sensitivity. The classifier maintained a high specificity in additional controls (98.3%), patients with chronic kidney (90.9%) and coronary artery (96.8%) diseases. Furthermore, in patients with FMD, the proteomic score was positively associated with radial wall thickness and wall cross-sectional area. In conclusion, a proteomic score has the potential to discriminate between patients with FMD and controls. If confirmed in a wider and more diverse cohort, these findings may pave the way for a noninvasive diagnostic test of FMD.

Metabolomics reveals elevated urinary excretion of collagen degradation and epithelial cell turnover products in irritable bowel syndrome patients.

INTRODUCTION: Irritable bowel syndrome (IBS), the most commonly diagnosed functional gastrointestinal (GI) disorder in developed countries, is characterized by chronic abdominal pain, and altered bowel habits. OBJECTIVES: Accurate and timely diagnosis is challenging as it relies on symptoms and an evolving set of exclusion criteria to distinguish it from other related GI disorders reflecting a complex etiology that remains poorly understood. Herein, nontargeted metabolite profiling of repeat urine specimens collected from a cohort of IBS patients (n = 42) was compared to healthy controls (n = 20) to gain insights into the underlying pathophysiology. METHODS: An integrated data workflow for characterization of the urine metabolome with stringent quality control was developed to authenticate reliably measured (CV < 30%) and frequently detected (> 75%) metabolites using multisegment injection-capillary electrophoresis-mass spectrometry. Complementary statistical methods were then used to rank differentially excreted urinary metabolites after normalization to osmolality that were subsequently identified by high resolution tandem mass spectrometry and their electrophoretic migration behavior. RESULTS: Our work revealed ten consistently elevated urinary metabolites in repeat samples collected from IBS patients at two different time points (q < 0.05 after age and Benjamini-Hochberg/FDR adjustment), which were associated with greater collagen degradation and intestinal mucosal turn-over processes likely due to low-grade inflammation. IBS-specific metabolites identified in urine included a series of hydroxylysine metabolites (O-glycosylgalactosyl-hydroxylysine, O-galactosyl-hydroxylysine, lysine), mannopyranosy-L-tryptophan, imidazole propionate, glutamine, serine, ornithine, dimethylglycine and dimethylguanosine. A major limitation in this retrospective case-control study was significant co-morbidity of IBS patients with other illnesses, including depression and prescribed medications as compared to healthy controls. CONCLUSION: This work provides new mechanistic insights into the pathophysiology of IBS while also offering a convenient way to monitor patient disease progression and treatment responses to therapy based on a panel of urinary metabolites that avoids invasive blood sampling, colonoscopy and/or tissue biopsies.

Association of kidney fibrosis with urinary peptides: a path towards non-invasive liquid biopsies?

Chronic kidney disease (CKD) is a prevalent cause of morbidity and mortality worldwide. A hallmark of CKD progression is renal fibrosis characterized by excessive accumulation of extracellular matrix (ECM) proteins. In this study, we aimed to investigate the correlation of the urinary proteome classifier CKD273 and individual urinary peptides with the degree of fibrosis. In total, 42 kidney biopsies and urine samples were examined. The percentage of fibrosis per total tissue area was assessed in Masson trichrome stained kidney tissues. The urinary proteome was analysed by capillary electrophoresis coupled to mass spectrometry. CKD273 displayed a significant and positive correlation with the degree of fibrosis (Rho = 0.430, P = 0.0044), while the routinely used parameters (glomerular filtration rate, urine albumin-to-creatinine ratio and urine protein-to-creatinine ratio) did not (Rho = -0.222; -0.137; -0.070 and P = 0.16; 0.39; 0.66, respectively). We identified seven fibrosis-associated peptides displaying a significant and negative correlation with the degree of fibrosis. All peptides were collagen fragments, suggesting that these may be causally related to the observed accumulation of ECM in the kidneys. CKD273 and specific peptides are significantly associated with kidney fibrosis; such an association could not be detected by other biomarkers for CKD. These non-invasive fibrosis-related biomarkers can potentially be implemented in future trials.

Diagnostic and prognostic role of urinary collagens in primary human bladder cancer.

Collagen type 4 alpha 1 (COL4A1) and collagen type 13 alpha 1 (COL13A1) produced by urothelial cancer cells support the vital oncogenic property of tumor invasion. We investigated the diagnostic and prognostic capability of COL4A1 and COL13A1 in voided urine and compared the observed values with those of fragments of cytokeratin-19 (CYFRA21-1), nuclear matrix protein 22 (NMP-22), and voided urine cytology in bladder cancer (BCa). We collected voided urine samples from 154 patients newly diagnosed with BCa, before surgery and from 61 control subjects. Protein levels of COL4A1, COL13A1, CYFRA21-1, and NMP-22 in urine supernatants were measured using enzyme-linked immunosorbent assays. Diagnostic performance and optimal cut-off values were determined by receiver operating characteristic analysis. Urine levels of COL4A1, COL13A1, the combined values of COL4A1 and COL13A1 (COL4A1 + COL13A1), and CYFRA21-1 were significantly elevated in urine from patients with BCa compared to the controls. Among these biomarkers, the optimal cut-off value of COL4A1 + COL13A1 at 1.33 ng/mL resulted in 57.4%, 83.7%, 56.1%, 80.7%, and 91.7% sensitivity for low-grade tumors, high-grade tumors, Ta, T1, and muscle invasive disease, respectively. We evaluated the prognostic value of preoperative urine levels in 130 non-muscle invasive BCa samples after the initial transurethral surgery. A high urinary COL4A1 + COL13A1 was found to be an independent risk factor for intravesical recurrence. Although these data need to be externally validated, urinary COL4A1 and COL13A1 could be a potential diagnostic and prognostic biomarker for BCa. This easy-to-use urinary signature identifies a subgroup of patients with a high probability of recurrence and progression in non-muscle invasive and muscle invasive BCa.

Alterations in urinary collagen peptides in lupus nephritis subjects correlate with renal dysfunction and renal histopathology.

BACKGROUND: The excessive accumulation of extracellular matrix (ECM) in the renal tubulointerstitium is a key component of chronic renal damage in lupus nephritis (LN) and a critical determinant of the disease progression to renal failure. Detection of fibrosis requires renal biopsy and is therefore limited by high risks associated with an invasive procedure. This study explores whether a unique LN urinary peptidome can be identified and whether LN-specific alteration reflects the underlying fibrogenic process of altered ECM turnover. METHOD: Urinary peptides were analyzed for 36 LN and 35 nonrenal systemic lupus erythematosus (SLE) subjects and 58 healthy volunteers (HVs). RESULTS: In total, 70 collagen and 230 noncollagen peptides were significantly changed between LN and nonrenal SLE and between LN and HV and defined as 'LN peptides'; 14 proteases associated with observed LN collagen peptides were identified and activities in 9 proteases were significantly different between LN and nonrenal SLE; 28 collagen peptides were correlated with at least one parameter of clinical renal dysfunction or histolopathology. CONCLUSION: Urinary peptidomic alterations likely reflect pathogenic pathways involving ECM turnover in LN kidneys and potentially could be developed as biomarkers to monitor renal disease progression.

Diastolic Left Ventricular Function in Relation to Urinary and Serum Collagen Biomarkers in a General Population.

Current knowledge on the pathogenesis of diastolic heart failure predominantly rests on case-control studies involving symptomatic patients with preserved ejection fraction and relying on invasive diagnostic procedures including endomyocardial biopsy. Our objective was to gain insight in serum and urinary biomarkers reflecting collagen turnover and associated with asymptomatic diastolic LV dysfunction. We randomly recruited 782 Flemish (51.3% women; 50.5 years). We assessed diastolic LV function from the early and late diastolic peak velocities of the transmitral blood flow and of the mitral annulus. By sequencing urinary peptides, we identified 70 urinary collagen fragments. In serum, we measured carboxyterminal propeptide of procollagen type 1 (PICP) as marker of collagen I synthesis and tissue inhibitor of matrix metalloproteinase type 1 (TIMP-1), an inhibitor of collagen-degrading enzymes. In multivariable-adjusted analyses with Bonferroni correction, we expressed effect sizes per 1-SD in urinary collagen I (uCI) or collagen III (uCIII) fragments. In relation to uCI fragments, e' decreased by 0.183 cm/s (95% confidence interval, 0.017 to 0.350; p = 0.025), whereas E/e' increased by 0.210 (0.067 to 0.353; p = 0.0012). E/e' decreased with uCIII by 0.168 (0.021 to 0.316; p = 0.018). Based on age-specific echocardiographic criteria, 182 participants (23.3%) had subclinical diastolic LV dysfunction. Partial least squares discriminant analysis contrasting normal vs. diastolic LV dysfunction confirmed the aforementioned associations with the uCI and uCIII fragments. PICP and TIMP-1 increased in relation to uCI (p<0.0001), whereas these serum markers decreased with uCIII (p≤0.0006). Diastolic LV dysfunction was associated with higher levels of TIMP-1 (653 vs. 696 ng/mL; p = 0.013). In a general population, the non-invasively assessed diastolic LV function correlated inversely with uCI and serum markers of collagen I deposition, but positively with uCIII. These observations generalise previous studies in patients to randomly recruited people, in whom diastolic LV function ranged from normal to subclinical impairment, but did not encompass overt diastolic heart failure.

Preoperative Urodynamic Parameters (Valsalva Leak Point Pressure and Maximum Urethral Closure Pressure), Urinary Collagen and Plasma Vitamin D Levels as Predictors of Mid Urethral Sling Surgery Outcome.

PURPOSE: To determine the best predictor of the mid urethral sling outcome we calculated the AUC of ROC curves of preoperative parameters, including Valsalva leak point pressure, maximum urethral closure pressure, urinary NTx (N-telopeptide of crosslinked type I collagen) and plasma vitamin D values (D2, D3 and D2 plus D3). MATERIALS AND METHODS: This was an ancillary study of TOMUS (Trial of Mid-urethral Slings) and the ValUE (Value of Urodynamics Evaluation) trial in which subjects underwent mid urethral sling surgery for stress urinary incontinence. Valsalva leak point pressure and maximum urethral closure pressure were measured in 427 subjects, whereas NTx, vitamin D2, vitamin D3 and vitamin D2 plus D3 levels were obtained from 150, 116, 115 and 116 subjects respectively. Outcome success was defined using identical outcome (subjective and objective) variables for all subjects. ROC curves with corresponding AUC values were compared. RESULTS: TOMUS and ValUE subjects were significantly different in age, body mass index, UDI (Urogenital Distress Inventory) scores. TOMUS subjects had a lower surgical success rate compared to ValUE subjects (66.3% vs 76.0%, p = 0.03). The AUC values of Valsalva leak point pressure, maximum urethral closure pressure, NTx, and vitamins D2, D3 and D2 plus D3 were 0.542, 0.561, 0.702, 0.627, 0.645 and 0.640, respectively. The AUC of NTx was significantly higher than the AUCs of Valsalva leak point pressure and maximum urethral closure pressure (p = 0.02 and 0.03, respectively). CONCLUSIONS: Urinary NTx was the best predictor of the mid urethral sling outcome. This test is not only noninvasive, it is also modifiable. Finding ideal modifiable risk factors prior to mid urethral sling surgery should be subject to future investigations.

Monoclonal antibody against macrophage colony-stimulating factor suppresses circulating monocytes and tissue macrophage function but does not alter cell infiltration/activation in cutaneous lesions or clinical outcomes in patients with cutaneous lupus erythematosus.

This study's objective was to assess the effects of PD-0360324, a fully human immunoglobulin G2 monoclonal antibody against macrophage colony-stimulating factor in cutaneous lupus erythematosus (CLE). Patients with active subacute CLE or discoid lupus erythematosus were randomized to receive 100 or 150 mg PD-0360324 or placebo via intravenous infusion every 2 weeks for 3 months. Blood and urine samples were obtained pre- and post-treatment to analyse pharmacokinetics and pharmacodynamic changes in CD14(+) CD16(+) monocytes, urinary N-terminal telopeptide (uNTX), alanine/aspartate aminotransferases (ALT/AST) and creatine kinase (CK); tissue biopsy samples were taken to evaluate macrophage populations and T cells using immunohistochemistry. Clinical efficacy assessments included the Cutaneous Lupus Erythematosus Disease Area and Severity Index (CLASI). Among 28 randomized/analysed patients, peak/trough plasma concentrations increased in a greater-than-dose-proportional manner with dose increases from 100 to 150 mg. Statistically significant differences were observed between active treatment and placebo groups in changes from baseline in CD14(+) CD16(+) cells, uNTX, ALT, AST and CK levels at most time-points. The numbers, density and activation states of tissue macrophages and T cells did not change from baseline to treatment end. No between-group differences were seen in CLASI. Patients receiving PD-0360324 reported significantly more adverse events than those receiving placebo, but no serious adverse events. In patients with CLE, 100 and 150 mg PD-0360324 every 2 weeks for 3 months suppressed a subset of circulating monocytes and altered activity of some tissue macrophages without affecting cell populations in CLE skin lesions or improving clinical end-points.

Stage-specific quantitative changes in renal and urinary proteome during the progression and development of streptozotocin-induced diabetic nephropathy in rats.

Diabetic nephropathy (DN) is a microvascular complication associated with diabetes causing slow deterioration of kidneys leading to end-stage renal disease. Timely intervention and diagnosis are crucial in order to ameliorate and halt the progression of DN. Current diagnosis of DN consists of urine assays for detection of microalbuminuria, which have inadequate specificity and sensitivity. Hence, there arises a need to discover stage-specific biomarkers which can aid in the early detection of DN and also in identifying the mechanisms underlying pathogenesis of DN. Therefore the present study was undertaken to identify the differentially expressed proteins in the urine and to examine the pattern of proteomic changes occurring in the rat kidneys during the course of progression of streptozotocin-induced model of DN in rats. Two-dimensional gel electrophoresis coupled to MALDI-TOF mass spectrometry was employed to identify the differentially expressed proteins under diabetic conditions. Among the identified proteins Calgranulin A and Calgranulin B appeared in the urinary proteome at the fourth week of induction of diabetes while we recorded a time-dependent decrease in the expression of major urinary protein (alpha 2u globulin) in the urine as well as kidneys of diabetic rats. Parallel monitoring of targeted proteomic changes in the renal proteome revealed an increase in histone H2B phosphorylation at serine14 along with a gradual decrease in Bcl-2 and MMP-13 expression during the course of progression and development of streptozotocin-induced DN.

Collagen peptides in urine: a new promising biomarker for the detection of colorectal liver metastases.

INTRODUCTION: For both patients and the outpatient clinic the frequent follow-up visits after a resection of colorectal cancer (CRC) are time consuming and due to large patient numbers expensive. Therefore it is important to develop an effective non-invasive test for the detection of colorectal liver metastasis (CRLM) which could be used outside the hospital. The urine proteome is known to provide detailed information for monitoring changes in the physiology of humans. Urine collection is non-invasive and urine naturally occurring peptides (NOPs) have the advantage of being easily accessible without labour-intensive sample preparation. These advantages make it potentially useful for a quick and reliable application in clinical settings. In this study, we will focus on the identification and validation of urine NOPs to discriminate patients with CRLM from healthy controls. MATERIALS AND METHODS: Urine samples were collected from 24 patients with CRLM and 25 healthy controls. In the first part of the study, samples were measured with a nano liquid chromatography (LC) system (Thermo Fisher Scientific, Germaring, Germany) coupled on-line to a hybrid linear ion trap/Orbitrap mass spectrometer (LTQ-Orbitrap-XL, Thermo Fisher Scientific, Bremen, Germany). A discovery set was used to construct the model and consecutively the validation set, being independent from the discovery set, to check the acquired model. From the peptides which were selected, multiple reaction monitoring (MRM's) were developed on a UPLC-MS/MS system. RESULTS: Seven peptides were selected and applied in a discriminant analysis a sensitivity of 84.6% and a specificity of 92.3% were established (Canonical correlation:0.797, Eigenvalue:1.744, F:4.49, p:0.005). The peptides AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGA P(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG were selected for further quantitative analysis which showed a sensitivity of 88% and a specificity of 88%. CONCLUSION: Urine proteomic analysis revealed two very promising peptides, both part from collagen type 1, AGPP(-OH)GEAGKP(-OH)GEQGVP(-OH)GDLGAP(-OH)GP and KGNSGEP(-OH)GAPGSKGDTGAKGEP(-OH)GPVG which could detect CRLM in a non-invasive manner.

Therapeutic effects of human mesenchymal stem cells in Wistar-Kyoto rats with anti-glomerular basement membrane glomerulonephritis.

INTRODUCTION: Multipotent mesenchymal stem cells (MSCs) have become a promising therapeutic approach in many clinical conditions. The hypothesis that MSCs can provide a potential therapy for human anti-glomerular basement membrane (GBM) glomerulonephritis (GN) was tested. METHODS: Nephrotoxic serum nephritis was induced in Wistar-Kyoto rats on day 0. Groups of animals were given either human MSCs (hMSCs, 3×10(6)) or vehicle by intravenous injection on day 4; all rats were sacrificed at either day 7 or day 13. RESULTS: Fluorescently labeled hMSCs were localized in glomeruli and tubulointerstitium 5 h after hMSC administration and persisted until 48 h, but hMSCs were barely detectable after 7 days. hMSC-treated rats had decreased kidney weight, proteinuria, and glomerular tuft area at each time point. The serum creatinine level and degree of glomerular crescent formation were decreased by hMSC treatment on day 13. ED1-positive macrophages, CD8-positive cells, and TUNEL-positive apoptotic cells in glomeruli were reduced by hMSC treatment on day 7, and this trend in apoptotic cells persisted to day 13. Renal cortical mRNA for TNF-α, IL-1ß, and IL-17, and the serum IL-17A level were decreased, whereas renal cortical mRNA for IL-4 and Foxp3 and the serum IL-10 level were increased in the MSC-treated group on day 7. Collagen types I and III and TGF-ß mRNA were decreased by hMSC treatment on day 13. CONCLUSION: The present results demonstrated that anti-inflammatory and immunomodulatory effects were involved in the mechanism of attenuating established experimental anti-GBM GN by hMSCs. These results suggest that hMSCs are a promising therapeutic candidate for the treatment of anti-GBM GN.

Non-sex cord-stromal ovarian tumors frequently produce and secrete estrogen in postmenopausal women: impact on bone metabolism and abnormal endometrial histology.

OBJECTIVE: We investigated whether the estrogen released from non-sex cord-stromal ovarian tumors has biological effects on bone and endometrial tissue in postmenopausal women. MATERIALS AND METHODS: A total of 132 patients with malignant (n = 112) or benign (n = 20) gynecological disease with normal postmenopausal ovaries were recruited as a control group (group C). The 75 patients with ovarian tumors were divided into 2 groups: group L (serum estradiol [E2] level of < 20 pg/ml; n = 42) and group H (serum E2 level of > 20 pg/ml; n = 33). The subjects' serum E2 levels, bone mineral density, urinary levels of the N-telopeptide of type I collagen, and serum levels of bone-specific alkaline phosphatase were measured before and after surgery. Histological examinations of the endometrium were carried out in the 75 patients with non-sex cord-stromal ovarian tumors. RESULTS: High serum E2 levels were detected in 33 (44%) of 75 patients with ovarian tumors. The serum E2 levels of group H were significantly decreased after surgery. In the other subjects, there were no significant differences between the serum E2 levels observed before and after surgery. The mean preoperative N-telopeptide of type I collagen and bone-specific alkaline phosphatase levels of group H were significantly lower than those of group C. The bone mineral density values of groups L and H were significantly decreased after surgery. Five (6.7%) of 75 patients with non-sex cord-stromal ovarian tumors displayed abnormal endometrial histology, including 4 patients with grade 1 endometrioid cancer. CONCLUSIONS: The E2 released from ovarian tumors might have biological effects on bone metabolism and the incidence of abnormal endometrial histology.

Changes in bone resorption across the menopause transition: effects of reproductive hormones, body size, and ethnicity.

OBJECTIVE: Our objective was to characterize changes in bone resorption in relation to the final menstrual period (FMP), reproductive hormones, body mass index (BMI), and ethnicity. METHODS: Urinary type I collagen N-telopeptide (NTX), estradiol, and FSH levels were measured annually for up to 8 years spanning the menopause transition in 918 African American, Chinese, Japanese, or Caucasian women. RESULTS: Urinary NTX began to increase sharply about 2 years before the FMP, reaching its peak level about 1 to 1.5 years after the FMP. NTX levels declined modestly from 2 to 6 years after the FMP but remained about 20% higher than before the menopause transition. The sharp rise in FSH occurred in conjunction with a sharp decline in estradiol and shortly after FSH levels began increasing rapidly. The mean increase in urinary NTX across the menopause transition was greatest in women with BMI <25 kg/m² and smallest in women with BMI >30 kg/m². Increases in NTX were greatest in Japanese women and smallest in African Americans. These differences were attenuated, but not eliminated, when analyses were adjusted for covariates, particularly BMI. SUMMARY: During the menopause transition, a decline in ovarian function beginning about 2 years before the FMP is followed by an increase in bone resorption and subsequently by bone loss. The magnitude of the increase in bone resorption is inversely associated with BMI. Ethnic differences in changes in bone resorption are attenuated, but not eliminated, by adjustment for BMI. Ethnic differences in BMI, and corresponding ethnic differences in bone resorption, appear to account for much of the ethnic variation in perimenopausal bone loss.

Ameliorative effect of Drynaria quercifolia (L.) J. Sm., an ethnomedicinal plant, in arthritic animals.

Drynaria quercifolia (L.) J. Sm., is an ethnomedicinal plant used widely in Tamil Nadu to treat arthritis. The present study was aimed to evaluate the traditional claim of D. quercifolia rhizome water extract in adjuvant induced arthritic animals. Anti-arthritic effect was studied by assessing the levels of lysosomal enzymes, protein bound carbohydrates, urinary degradative collagen and serum cytokines on control and adjuvant induced arthritis. The paw swelling and body weight were also analyzed. The levels of ROS and lysosomal enzymes in neutrophils of control and adjuvant induced animals were also estimated. D. quercifolia rhizome water extract at doses of 100 and 200mg/kg reduced the paw thickness and elevated the mean body weight of arthritic rats. The treatment with extract showed a significant reduction in the levels of plasma and liver lysosomal enzymes as well as protein bound carbohydrates and urinary degradative collagen levels. The treatment reduced the levels of ROS and lysosomal enzymes in neutrophils significantly. The significant reduction in the levels of serum pro-inflammatory cytokines (TNF-α and IL-1ß) and the increment in the levels of anti-inflammatory cytokine (IL-10) were also observed by the treatment. The present study supports the traditional claim of using D. quercifolia to treat rheumatism.

Therapeutic effect of Saraca asoca (Roxb.) Wilde on lysosomal enzymes and collagen metabolism in adjuvant induced arthritis.

Rheumatoid arthritis is a chronic, progressive and systemic inflammatory disorder mainly affecting the synovial joints. In the present study, we evaluated the anti-arthritic effect of the methanol extract of Saraca asoca (Roxb.) Wilde., (Fabaceae) on adjuvant induced arthritis by assessing paw swelling, body weight, the levels of lysosomal enzymes, protein bound carbohydrates, serum cytokines, urinary collagen and histopathology of joints. It was found that S. asoca methanol extract at doses of 50, 100 and 200 mg/kg reduced the paw thickness and elevated the mean body weight of arthritic rats. The treatment of S. asoca showed a significant reduction in the levels of both plasma and liver lysosomal enzymes. The protein bound carbohydrates and urinary collagen contents were also decreased at a significant level by the treatment of S. asoca methanol extract. The histopathological study of the joints showed the anti-arthritic property of S. asoca which nearly normalized the histological architecture of the joints. Further, we established the anti-arthritic activity of S. asoca methanol extract by measuring the levels of cytokines in both arthritic and treated rats. The treatment of S. asoca reduced the levels of pro-inflammatory cytokines. In conclusion, S. asoca methanol extract was capable of ameliorating the conditions of arthritis in adjuvant induced arthritic rats.

Deletion of soluble epoxide hydrolase gene improves renal endothelial function and reduces renal inflammation and injury in streptozotocin-induced type 1 diabetes.

Studies suggest that soluble epoxide hydrolase (sEH) inhibition reduces end-organ damage in cardiovascular diseases. We hypothesize that sEH gene (Ephx2) knockout (KO) improves endothelial function and reduces renal injury in streptozotocin-induced diabetes. After 6 wk of diabetes, afferent arteriolar relaxation to acetylcholine was impaired in diabetic wild-type (WT) mice, as the maximum relaxation was 72% of baseline diameter in the WT but only 31% in the diabetic mice. Ephx2 KO improved afferent arteriolar relaxation to acetylcholine in diabetes as maximum relaxation was 58%. Urinary monocyte chemoattractant protein-1 (MCP-1) excretion significantly increased in diabetic WT mice compared with control (868 ± 195 vs. 31.5 ± 7 pg/day), and this increase was attenuated in diabetic Ephx2 KO mice (420 ± 98 pg/day). The renal phospho-IKK-to-IKK ratio and nuclear factor-κB were significantly decreased, and hemeoxygenase-1 (HO-1) expression increased in diabetic Ephx2 KO compared with diabetic WT mice. Renal NADPH oxidase and urinary thiobarbituric acid reactive substances excretion were reduced in diabetic Ephx2 KO compared with diabetic WT mice. Albuminuria was also elevated in diabetic WT mice compared with control (170 ± 43 vs. 37 ± 13 µg/day), and Ephx2 KO reduced this elevation (50 ± 15 µg/day). Inhibition of sEH using trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (tAUCB) also reduced renal inflammation and injury in diabetic WT mice. Furthermore, inhibition of HO with stannous mesoporphyrin negated the reno-protective effects of tAUCB or Ephx2 KO during diabetes. These data demonstrate that Ephx2 KO improves endothelial function and reduces renal injury during diabetes. Additionally, our data also suggest that activation of HO-1 contributes to improved renal injury in diabetic Ephx2 KO mice.

Bone resorption in syndromes of the Ras/MAPK pathway.

Disorders of the Ras/mitogen-activated protein kinase (MAPK) pathway have an overlapping skeletal phenotype (e.g. scoliosis, osteopenia). The Ras proteins regulate cell proliferation and differentiation and neurofibromatosis type 1 (NF1) individuals have osteoclast hyperactivity and increased bone resorption as measured by urine pyridinium crosslinks [pyridinoline (Pyd) and deoxypyridinoline (Dpd)]. Pyd and Dpd are hydroxylysine-derived crosslinks of collagen found in bone and cartilage and excreted in the urine. Dpd is most abundant in bone. The aim of this study was to evaluate if other syndromes of the Ras/MAPK pathway have increased bone resorption, which may impact the skeletal phenotype. Participants were individuals with Noonan syndrome (n = 14), Costello syndrome (n = 21), and cardiofaciocutaneous (CFC) syndrome (n = 14). Pyridinium crosslinks from two consecutive first morning urines were extracted after acid hydrolysis and analyzed by high performance liquid chromatography. Three separate analyses of covariance were performed to compare Pyd, Dpd, and Dpd/Pyd ratio of each group to controls after controlling for age. Data were compared to 99 healthy controls. The Dpd and the Dpd/Pyd ratio were elevated (p < 0.0001) in all three conditions compared to controls suggesting that collagen degradation was predominantly from bone. The data suggest that the Ras/MAPK signal transduction pathway is important in bone homeostasis.

Collagen XXIII: a potential biomarker for the detection of primary and recurrent non-small cell lung cancer.

BACKGROUND: Collagen XXIII is a transmembrane collagen previously shown to be upregulated in metastatic prostate cancer. The purpose of this study was to determine the protein expression of collagen XXIII in tumor tissues from a variety of cancers and to assess the utility of collagen XXIII as a biomarker for non-small cell lung cancer (NSCLC). METHODS: A multicancer tissue microarray was used for the immunohistochemical examination of collagen XXIII protein expression in a variety of cancers. Subsequently, collagen XXIII expression was analyzed in three separate cohorts using tissue microarrays with representative tumor and control lung tissues from NSCLC patients. In addition, NSCLC patient urine samples were analyzed for the presence of collagen XXIII through Western blot. RESULTS: Collagen XXIII was present in tissue samples from a variety of cancers. Within lung cancer tissues, collagen XXIII staining was enriched in NSCLC subtypes. Collagen XXIII was present in 294 of 333 (88%) lung adenocarcinomas and 97 of 133 (73%) squamous cell carcinomas. In urine, collagen XXIII was present in 23 of 29 (79%) NSCLC patient samples but only in 15 of 54 (28%) control samples. High collagen XXIII staining intensity correlated with shorter recurrence-free survival in NSCLC patients. CONCLUSIONS: We show the capability of collagen XXIII as a tissue and urinary biomarker for NSCLC, in which positivity in tissue or urine significantly correlates with the presence of NSCLC and high staining intensity is a significant recurrence predictor. IMPACT: Inclusion of collagen XXIII in a tissue- or urine-based cancer biomarker panel could inform NSCLC patient treatment decisions.