Universal nanocomposite coating with antifouling and redox capabilities for electrochemical affinity biosensing in complex biological fluids.

Electrochemical affinity biosensors have the potential to facilitate the development of multiplexed point-of-care diagnostics in complex biological fluids. However, their commercial viability has been hindered by challenges such as electrode biofouling and the lack of inherent redox properties. To address this unmet need, we have developed a universal nanocomposite coating which is unique in its ability to not only allow oriented conjugation of the biorecognition element but also specific detection directly in complex biological fluids like serum and urine owing to its built-in antifouling and redox capabilities, thus improving suitability for point of care testing. This multifunctional coating comprises a 3D porous crosslinked bovine serum albumin matrix for oriented conjugation and antifouling properties with embedded graphene nanosheets modified with amino-ferrocene for enhanced conductivity and mediator-free biosensing. The coating showed minimal signal degradation despite prolonged exposure to 1% bovine serum albumin, artificial urine and untreated human serum for up to 30 days. To demonstrate its utility, we fabricated and tested proof-of-concept electrochemical immunosensors for bladder cancer protein biomarkers, specifically interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF). The practical feasibility was highlighted by the excellent sensitivity and specificity observed for IL-8 and VEGF with a limit of detection of 41 pg mL-1 and 67 pg mL-1, respectively. Consequently, this universal nanocomposite-based electrochemical biosensing platform can be extended to the point of care testing of a broad spectrum of biomarkers present in complex biological fluids, thus enabling reliable and early diagnostics.

Urinary interleukin-9 in youth with type 1 diabetes mellitus.

AIMS: Interleukin-9 (IL-9) attenuates podocyte injury in experimental kidney disease, but its role in diabetic nephropathy is unknown. We sought to relate urinary IL-9 levels to the release of podocyte-derived extracellular vesicles (EVs) in youth with type 1 diabetes. We related urinary IL-9 levels to clinical variables and studied interactions between urinary IL-9, vascular endothelial growth factor (VEGF), tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) on urinary albumin/creatinine ratio (ACR) a functional measure of podocyte injury. METHODS: We performed an analysis of urine samples and clinical data from a cohort of youth with type 1 diabetes (n = 53). Cytokines were measured using a Luminex platform (Eve Technologies), and nanoscale flow cytometry was employed to quantify urinary podocyte-derived EVs. All urinary measures were normalized to urinary creatinine. RESULTS: Mean age was 14.7 ± 1.6 years, and the mean time from diagnosis was 6.7 ± 2.9 years. Mean HbA1c was 70.3 ± 13.9 mmol/mol, mean ACR was 1.3 ± 1.9 mg/mmol, and mean eGFR was 140.3 ± 32.6 ml/min/1.73 m2. IL-9 was inversely related to podocyte EVs (r = - 0.56, p = 0.003). IL-9 was also inversely related to blood glucose, HbA1C and eGFR (r = - 0.44, p = 0.002; r = - 0.41, p = 0.003; r = - 0.49, p < 0.001, respectively) and positively correlated with systolic BP (r = 0.30, p = 0.04). There was a significant interaction between IL-9, EVs and ACR (p = 0.0143), and the relationship between IL-9 and ACR depended on VEGF (p = 0.0083), TNFα (p = 0.0231) and IL-6 levels (p = 0.0178). CONCLUSIONS: IL-9 is associated with podocyte injury in early type 1 diabetes, and there are complex interactions between urinary IL-9, inflammatory cytokines and ACR.

Suramin enhances the urinary excretion of VEGF-A in normoglycemic and streptozotocin-induced diabetic rats.

BACKGROUND: Vascular endothelial growth factor A (VEGF-A) and P2-receptors (P2Rs) are involved in the pathogenesis of diabetic nephropathy. The processing of VEGF-A by matrix metalloproteinases (MMP) regulates its bioavailability. Since the ATP-induced release of MMP-9 is mediated by P2Rs, we investigated the effect of suramin on VEGF-A excretion in urine and the urinary activity of total MMP and MMP-9. METHODS: The effect of suramin (10 mg/kg, ip) on VEGF-A concentration in serum and its excretion in urine was investigated in streptozotocin (STZ)-induced diabetic rats over a 21-day period. The rats received suramin 7 and 14 days after a single STZ injection (65 mg/kg, ip). A 24-h collection of urine was performed on the day preceding the administration of STZ and the first administration of suramin and on the day before the end of the experiment. The VEGF-A in serum and urine, albumin in urine, and total activity of MMP and MMP-9 in urine were measured using immunoassays. RESULTS: Diabetic rats are characterized by a sixfold higher urinary excretion of VEGF-A. Suramin potentiates VEGF-A urinary excretion by 36% (p = 0.046) in non-diabetic and by 75% (p = 0.0322) in diabetic rats but it did not affect VEGF-A concentration in the serum of non-diabetic and diabetic rats. Urinary albumin excretion as well as total MMP and MMP-9 activity was increased in diabetic rats, but these parameters were not affected by suramin. CONCLUSION: Suramin increases the urinary excretion of VEGF-A in normoglycemia and hyperglycaemia, possibly without the involvement of MMP-9. Suramin may be used as a pharmacological tool enhancing VEGF-A urinary secretion.

Circulating Vascular Endothelial Growth Factor and Soluble fms-Like Tyrosine Kinase-1 as Biomarkers for Endometrial Remodeling Across the Menstrual Cycle.

OBJECTIVE: To characterize variation in circulating vascular endothelial growth factor (VEGF) and its receptor, soluble fms-like tyrosine kinase-1 (sFLT-1), across the menstrual cycle in normal ovulating women in relation to reproductive hormones to identify the utility of VEGF and sFLT-1 as peripheral biomarkers of endometrial remodeling. METHODS: Ninety-six healthy, regularly menstruating ovulatory women, aged 18-44 years, enrolled in the BioCycle Study, a prospective cohort study at a U.S. academic research center. Vascular endothelial growth factor and sFLT-1 were measured in concurrently collected plasma, serum, and urine up to eight times across a single cycle. Reproductive hormones were measured in serum. Mean concentrations of VEGF and sFLT-1 were compared across phases of the cycle, and correlations between specimen types were calculated. Harmonic models estimated associations between VEGF and sFLT-1 and characteristics of hormonal patterns. RESULTS: No variation in VEGF or sFLT-1 levels were detected over the menstrual cycle. Median (25th percentile, 75th percentile) concentrations of VEGF during the menstrual cycle were 31.2 pg/mL (24.1, 56.9) in plasma, 194.1 pg/mL (125.4, 350.2) in serum, and 101.7 pg/mL (64.2, 165.8) in urine. Plasma and serum measures were consistently correlated, whereas urinary measures were not. Vascular endothelial growth factor was not consistently associated with reproductive hormone concentrations, although sFLT-1 was associated with higher mean and amplitude of estradiol. CONCLUSION: Circulating VEGF and sFLT-1 did not vary across the menstrual cycle and therefore are unlikely to be useful peripheral biomarkers of endometrial changes across the menstrual cycle. For studies measuring circulating VEGF for other reasons, plasma may be the preferred medium and timing to menstrual cycle phase need not be considered for reproductive-age women.

Relationship of Bladder Pain With Clinical and Urinary Markers of Neuroinflammation in Women With Urinary Urgency Without Urinary Incontinence.

PURPOSE: The pathogenesis of bladder pain is poorly understood. Our hypothesis is that in women with urinary urgency without incontinence, bladder pain is associated with the presence of neurogenic inflammation in the bladder wall and neuroinflammatory biomarkers in the urine. METHODS: We conducted a prospective cross-sectional study of women with urinary urgency without incontinence. Urinary symptoms were measured using Female Genitourinary Pain Index. Neuropathic pain, a clinical biomarker of neuroinflammation, was measured using the PainDETECT questionnaire. Inflammatory neuropeptides measured in the urine included nerve growth factor (NGF), brain-derived neurotrophic factor, vascular endothelial growth factor, and osteopontin. Neuropathic pain scores and urinary neuropeptide levels were compared between patients with and without bladder pain using univariable and multivariable analyses. RESULTS: In 101 women with urinary urgency without incontinence, 62 (61%) were in the bladder pain group (visual analog scale score, ≤ 3), whereas 39 (39%) were in the no bladder pain group. Urinary symptom scores (5.0 ± 3.1 versus 3.5 ± 2.4, P < 0.001) and neuropathic pain scores (13.3 ± 8.6 vs 5.1 ± 4.8, P < 0.001) were significantly higher for the bladder pain group than for the no bladder pain group. On multivariable analysis after controlling for age, body mass index, and severity of urinary urgency, bladder pain score was significantly associated with elevated urinary levels of vascular endothelial growth factor (P = 0.04) and osteopontin (P = 0.02), whereas the neuropathic pain score was significantly associated with an increased NGF level (P = 0.03). CONCLUSIONS: In women with urinary urgency without incontinence, bladder pain is associated with the presence of clinical and urinary biomarkers of neuroinflammation.

The Effect of Hyperfiltration on Kidney Function in Living Donor Kidney Transplantation: A Prospective Cohort Study.

BACKGROUND: living kidney donation is a safe medical procedure. Kidney function after donation is crucial for donors' health and quality of life. Kidney hyperfiltration is a compensatory mechanism, which will preserve kidney function after unilateral nephrectomy. The number of studies regarding hyperfiltration in living kidney donors is limited. Our study aimed to explain kidney hyperfiltration mechanism and evaluate its effect on the kidney function within 30 days after surgery. METHODS: our study was a prospective cohort study with 46 living-kidney donors participating in the study between April and December 2019. We evaluated main outcomes, the 30-day post-surgery kidney function, which was evaluated by calculating estimated glomerular filtration rate (eGFR) and Urinary Albumin to Creatinine Ratio (ACR). The subjects were categorized into two groups based on their 30-day outcomes, which were the adaptive (eGFR > 60 mL/min/1.73 m2 and/or ACR > 30 mg/g) and maladaptive (eGFR < 60 mL/min/1.73 m2 and/or ACR > 30 mg/g) groups. A series of evaluation including calculating the renal arterial resistive index (RI) and measuring urinary vascular endothelial growth factor (VEGF), neutrophil gelatinase-associated lipocalin (NGAL), and heparan sulfate (HS) levels were performed before surgery and serially until 30 days after surgery. Multivariate analysis with adjustments for confounding factors was done. RESULTS: forty donors were included and mostly were female (67.5%). The average age and body mass index (BMI) were 45.85 (SD 9.74) years old and 24.36 (SD 3.73) kg/m2, respectively. Nineteen donors (47.5%) had maladaptive hyperfiltration outcomes. The hyperfiltration process was demonstrated by significant changes in renal arterial RI, urinary VEGF, NGAL, and HS levels (p<0.005). There was no significant difference regarding RI, urinary VEGF, NGAL, and HS levels between both groups. Several confounding factors (BMI over 25 kg/m2, familial relationship, age over 40 years old, and arterial stiffness) were significantly influenced by kidney hyperfiltration and outcomes (p<0.05). CONCLUSION: the hyperfiltration process does not affect the 30-day post-nephrectomy kidney function of the donors. Several other factors may influence the hyperfiltration process and kidney function. Further study is necessary to evaluate kidney function and its other related variables with a longer period of time study duration.

Repeated intravesical injections of platelet-rich plasma improve symptoms and alter urinary functional proteins in patients with refractory interstitial cystitis.

Repeated intravesical injections of autologous platelet-rich plasma (PRP) have been shown to improve symptoms in patients with interstitial cystitis/bladder pain syndrome (IC/BPS); however, there is a paucity of objective evidence of the effectiveness of this therapy. In this study, we investigated the changes in urinary markers after PRP treatment. Forty patients with IC/BPS who were refractory to conventional therapy received four injections of PRP at monthly intervals; 10 mL PRP solution with 2.5 times the peripheral blood platelet concentration was used. Urine levels of thirteen functional proteins, growth factors, and cytokines were assessed at baseline and at the 4th PRP injection. The clinical parameters included visual analog scale (VAS) pain score, daily urinary frequency, nocturia episodes, functional bladder capacity, and global response assessment (GRA). The GRA and symptom score significantly decreased post-treatment. In patients with GRA ≥ 2, the success rates at 1 month and at 3 months after the 4th PRP injection were 70.6% and 76.7%, respectively. The VAS pain score, frequency, and nocturia showed a significant decrease (all p < 0.05). Urinary levels of nerve growth factor, matrix metalloproteinase-13, and vascular endothelial growth factor significantly decreased post-treatment (p = 0.043, p = 0.02, and p = 0.000, respectively); platelet-derived growth factor-AB showed a significant increase (p = 0.004) at the 4th PRP treatment compared with baseline. In this study, repeated intravesical PRP injections provided significant symptom improvement in IC/BPS patients with concomitant changes in the related biomarker levels.Trial registration: ClinicalTrial.gov: NCT03104361; IRB: TCGH 105-48-A.

Simultaneous Sensing of Multiple Cancer Biomarkers by a Single DNA Nanoprobe in a Nanopore.

Both vascular endothelial growth factor (VEGF) and matrix metallopeptidase-9 (MMP-9) are key biomarkers in tumor angiogenesis. Determination of the overexpression of the two biomarkers would provide valuable information on the progression of tumor growth and metastasis, but their simultaneous quantification by a single probe is unprecedented. Here, we develop a triplex DNA-based nanoprobe for simultaneously quantifying VEGF and MMP-9 using an α-hemolysin nanopore. A DNA aptamer is used as the triplex molecular beacon (tMB) loop to bind VEGF, and a stem-forming oligonucleotide modified with a short peptide is used to recognize MMP-9. The sequential presence of VEGF and MMP-9 could also be identified by different patterns of current events. Besides, the characteristic current events generated by the DNA probe possess pH-dependent patterns that can be used to reflect the environmental pH. Success in the construction of such DNA nanoprobes will greatly facilitate the investigation of the mechanisms of different tumor angiogenesis processes and provide a useful approach for cancer diagnosis.

Panel of novel urine biomarkers for incident microalbuminuria in people with type 2 diabetes mellitus.

AIM: The need to identify novel biomarkers for early diagnosis and treatment of diabetic nephropathy is widely recognized. However, only a few studies have investigated the association between biomarkers and the onset of albuminuria. In this study, we aimed to investigate a panel of biomarkers suitable for predicting microalbuminuria. METHODS: Some 346 Japanese people with type 2 diabetes exhibiting normoalbuminuria were studied. The endpoint was defined as the onset of microalbuminuria. Thirty biomarkers were selected from among urinary biomarkers described in previous studies. A panel of biomarkers was selected using least absolute shrinkage and selection operator (LASSO). The prognostic performance of the developed panel was evaluated. RESULTS: During a mean follow-up of 6.2 years, 45 people progressed to microalbuminuria. A composite panel of six biomarkers (monocyte chemoattractant protein-1, osteopontin, soluble human tumour necrosis factor receptor-1, tenascin C, vascular endothelial growth factor-A and kidney injury molecule-1) was developed using LASSO. Compared with the basal model comprising estimated GFR and urinary albumin-to-creatinine ratio, addition of these six biomarkers significantly increased the overall C index from 0.773 to 0.824 (P = 0.019). Net reclassification improvement and integrated discrimination improvement were estimated to be 0.412 (P = 0.049) and 0.040 (P = 0.040), respectively. Decision curve analysis also showed that the model with the six novel biomarkers had a better prognostic value for predicting the onset of microalbuminuria. The optimism was moderate or negligible according to measures. CONCLUSIONS: The panel consisting of six novel urinary biomarkers effectively predicted incident microalbuminuria in people with type 2 diabetes.

Urinary biomarkers of latent inflammation and fibrosis in children with vesicoureteral reflux.

PURPOSE: To investigate the urinary levels of TGF-ß1, VEGF, and MCP-1 as potential biomarkers of latent inflammation and fibrosis in the kidney before and 6 months after correction of vesicoureteral reflux (VUR) in children. METHODS: A total of 88 patients (mean age 26 months) with VUR were divided into three groups: group A-patients with grades II-III VUR, conservative treatment; group B-patients with grades III-V VUR, endoscopic correction of VUR; group C-patients with grades III-V VUR, ureteral reimplantation after failed endoscopic correction. Control group included 20 healthy children. Biomarker levels were measured by ELISA. 99mTc-DMSA scintigraphy and renal histology were performed if possible. RESULTS: At admission, TGF-ß1 was close to control in all study groups, VEGF increased with severity of the disease, and MCP-1 increased in group C. Six months after correction of VUR, despite clinical and laboratory improvement, TGF-ß1 and MCP-1 increased while VEGF decreased compared to the admission values in all groups; no amelioration of renal scarring was detected either by 99mTc-DMSA scintigraphy or renal histology. CONCLUSION: The results support our hypothesis that successful correction of VUR is not sufficient to stop or reduce the latent inflammatory and fibrotic processes that have already started in the kidney regardless of the reflux grade and treatment option. Measuring the urinary levels of TGF-ß1, VEGF, and MCP-1 may aid in the development of non-invasive, pathophysiologically relevant approach to diagnosis and monitoring of kidney injury and fibrosis in children with VUR.

Soluble TNF-R1, VEGF and other cytokines as markers of disease activity in systemic lupus erythematosus and lupus nephritis.

BACKGROUND: Current non-invasive methods of assessing disease activity in systemic lupus erythematosus (SLE) are of limited sensitivity and specificity. Testing includes acute phase markers, autoantibodies and complement levels. Although measurements of dsDNA antibodies and complement C3/C4 levels are routine, they remain of limited value. Improved blood and urine markers may help in early detection of flare, distinction between flare and chronic damage, and monitoring response to therapy. METHODS: A total of 87 patients with SLE were tested for the following cytokines in serum and urine: monocyte chemoattractant protein 1 (MCP-1), regulated upon activation, normal T cell expressed and secreted (RANTES), soluble tumour necrosis factor receptor 1 (sTNF-R1), interferon-inducible protein 10 (IP-10), monocyte inhibitory protein 1α (MIP-1α) and vascular endothelial growth factor (VEGF). Patients attending the Lupus Unit at St Thomas' Hospital, London, UK were divided into active lupus nephritis (LN), inactive LN and non-renal SLE groups based on their renal pathology and SLE disease activity index (SLEDAI). Cytokine testing was performed using the FIDIS multiplex bead assay. RESULTS: The mean level of serum sTNF-R1 was higher in the active LN group compared with both inactive LN and non-renal SLE groups ( p < 0.001). For urine measurements there were significant differences between active LN and non-renal SLE for VEGF ( p = 0.016), after statistical correction for multiple testing. Both urinary and serum sTNF-R1 and IP-10 levels correlated with SLEDAI scores ( p < 0.001), while serum VEGF correlated weakly with SLEDAI ( p = 0.025). The optimum combination for differentiating active from inactive LN patients was serum VEGF, sTNF-R1, MCP-1 and glomerular filtration rate plus urinary sTNF-R1 and protein-creatinine ratio. CONCLUSION: These results indicate that for active LN, sTNF-R1 could be a useful serum cytokine marker, with potential for VEGF in the urine. This study has confirmed the ability of the multiplex bead technique to detect cytokines in a good analytical range, including very low and high levels, in both serum and urine. Combining serum and urine markers provided additional sensitivity in distinguishing active from inactive LN.

Flexible Biosensors for the Impedimetric Detection of Protein Targets Using Silk-Conductive Polymer Biocomposites.

To expand the applications of flexible biosensors in point-of-care healthcare applications beyond monitoring of biophysical parameters, it is important to devise strategies for the detection of various proteins and biomarkers. Here, we demonstrate a flexible, fully organic, biodegradable, label-free impedimetric biosensor for the critical biomarker, vascular endothelial growth factor (VEGF). This biosensor was constructed by photolithographically patterning a conducting ink consisting of a photoreactive silk sericin coupled with a conducting polymer. These functional electrodes are printed on flexible fibroin substrates that are controllably thick and can be free-standing, or conform to soft surfaces. Detection was accomplished via the antibody to VEGF which was immobilized within the conducting matrix. The results indicated that the developed flexible biosensor was highly sensitive and selective to the target protein, even in challenging biofluids such as human serum. The biosensors themselves are biocompatible and degradable. Through this work, the developed flexible biosensor based on a simple and label-free strategy can find practical applications in the monitoring of wound healing or early disease diagnosis.

Altered Angiogenic Growth Factors in Urine of Prostate Cancer Survivors With Radiation History and Radiation Cystitis.

OBJECTIVE: To determine if the vascular damage in bladders of prostate cancer (PCa) survivors with radiation cystitis can be detected through altered angiogenic growth factors in urine. METHODS: Urine samples from PCa survivors with a history of external beam radiation therapy were tested for a panel of angiogenic growth factors by Luminex assay. Urine creatinine levels were measured through high performance liquid chromatography. Through a patient survey, data on patient demographics, radiation history, and urinary symptoms were collected. RESULTS: Hepatocyte growth factor (HGF), placental growth factor (PlGF), and vascular endothelial growth factor (VEGF) were altered in urine of PCa survivors with a history of radiation therapy. HGF and PlGF were elevated in response to irradiation, while VEGF had a decreasing trend. Within the irradiated population, HGF was also increased in patients diagnosed with radiation cystitis and patients with hematuria. PlGF and VEGF were only increased in the first year postirradiation, and VEGF was elevated in patients with hematuria. Finally, creatinine levels were increased in PCa survivors with a history of radiation therapy. CONCLUSION: Radiation cystitis is a debilitating bladder condition that cancer survivors are at risk of developing after pelvic radiation. In this study, we identified 3 pro-angiogenic factors that may be urine biomarkers and, if validated in future studies, could indicate new strategy approaches to treat radiation cystitis.

Relationship between circulating vascular endothelial growth factor and its soluble receptor in patients with hemorrhagic fever with renal syndrome.

Hemorrhagic fever with renal syndrome (HFRS) is characterized by endothelial dysfunction with capillary leakage without obvious cytopathology in the capillary endothelium. The aim of the study was to analyze the kinetics of vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-2) in HFRS patients infected with Dobrava (DOBV) or Puumala virus (PUUV). VEGF and sVEGFR-2 levels were measured in daily plasma and urine samples of 73 patients with HFRS (58 with PUUV, 15 with DOBV) and evaluated in relation to clinical and laboratory variables. In comparison with the healthy controls, initial samples (obtained in the first week of illness) from patients with HFRS had higher plasma and urine VEGF levels, whereas sVEGFR-2 levels were lower in plasma but higher in urine. VEGF levels did not differ in relation to hantavirus species, viral load, or the severity of HFRS. The comparison of VEGF dynamics in plasma and urine showed the pronounced secretion of VEGF in urine. Significant correlations were found between daily VEGF/sVEGFR-2 levels and platelet counts, as well as with diuresis: the correlations were positive for plasma VEGF/sVEGFR-2 levels and negative for urine levels. In addition, patients with hemorrhagic manifestations had very high plasma and urine VEGF, together with high urine sVEGFR-2. Measuring the local secretion of sVEGFR-2 in urine might be a useful biomarker for identifying HFRS patients who will progress to severe disease.

Early pregnancy protein multiplex screening reflects circulating and urinary divergences associated with the development of preeclampsia.

BACKGROUND: Preeclampsia, a pregnancy disorder characterized by hypertension and proteinuria, represents the leading cause of fetal and maternal morbidity and mortality in developing countries. The identification of novel and accurate biomarkers that are predictive of preeclampsia is necessary to improve the prognosis of patients with preeclampsia. OBJECTIVE: To evaluate the preeclampsia predictive value of 34 angiogenic-related proteins. METHODS: We performed a nested cohort case-control study of pregnant women. The profile of the 34 proteins was evaluated at 12, 16, and 20 gestational weeks (GWs), using urine/plasma from 16 women who developed preeclampsia and 20 normotensive pregnant controls by Bio-Plex ProTM Human Cancer Biomarker Panels 1 and 2. RESULTS: The urine concentration of soluble epidermal growth factor receptor (sEGFR), hepatocyte growth factor (HGF), angiopoietin-2 (ANG-2), endoglin (ENG), soluble fas ligand (sFASL), interleukin 6 (IL-6), placental growth factor (PLGF), and vascular endothelial growth factor A (VEGF-A) at 12 GW, prolactin (PRL), ANG-2, transforming growth factor alpha (TGF-α), and VEGF-A at 16 GW, and soluble IL-6 receptor alpha (sIL-6Rα), ANG-2 and sFASL at 20 GW, were different between groups (p < 0.05). The concentration cut-off values calculated in this study for the mentioned proteins, predicted an increased risk to developing preeclampsia in a range of 3.8-29.8 times in the study population. CONCLUSION: The proteins sEGFR, HGF, ANG-2, sFASL, IL-6, PLGF, VEGF-A, PRL, TGF-α FGF-b, sHER2/Neu sIL-6Rα, ENG, uPA, and insulin-like growth factor binding protein 1 (IGFBP-1), were predictive of the development of preeclampsia and their use as markers for this disease should be considered.

Investigating Colorimetric Protein Array Assay Schemes for Detection of Recurrence of Bladder Cancer.

A colorimetric microarray for the multiplexed detection of recurrence of bladder cancer including protein markers interleukin-8 (IL8), decorin (DCN), and vascular endothelial growth factor (VEGF) was established to enable easy and cheap read-out by a simple office scanner paving the way for quick therapy monitoring at doctors' offices. The chip is based on the principle of a sandwich immunoassay and was optimized prior to multiplexing using IL8 as a model marker. Six different colorimetric assay formats were evaluated using a detection antibody (dAB) labeled with (I) gold (Au) nanoparticles (NPs), (II) carbon NPs, (III) oxidized carbon NPs, and a biotinylated dAB in combination with (IV) neutravidin-carbon, (V) streptavidin (strp)-gold, and (VI) strp-horseradish peroxidase (HRP). Assay Format (III) worked best for NP-based detection and showed a low background while the enzymatic approach, using 3,3',5,5'-tetramethylbenzidine (TMB) substrate, led to the most intense signals with good reproducibility. Both assay formats showed consistent spot morphology as well as detection limits lower than 15 ng/L IL8 and were thus applied for the multiplexed detection of IL8, DCN, and VEGF in synthetic urine. Colorimetric detection in urine (1:3) yields reaction signals and measurement ranges well comparable with detection in the assay buffer, as well as excellent data reproducibility as indicated by the coefficient of variation (CV 5-9%).

Comparison of inflammatory urine markers in patients with interstitial cystitis and overactive bladder.

INTRODUCTION AND HYPOTHESIS: Chronic inflammatory conditions seem to be a shared characteristic in patients with interstitial cystitis (IC) and overactive bladder (OAB). Thus, we measured 40 inflammatory urine markers in IC patients with or without Hunner's lesions (HIC and NHIC respectively) and OAB patients. METHODS: Urine was collected from consecutive HIC patients, NHIC patients, and age and gender-matched OAB patients with no history of IC, recurrent urinary tract infection or bladder cancer. The diagnosis of IC was based on the Asian IC guideline criteria. A representative 40 inflammatory growth factors, cytokines, and chemokines in urine were measured using a MILLIPLEX immunoassay kit. Statistical differences in these markers among the groups were determined by nonparametric ANOVA followed by multiple comparison test. The diagnostic efficiency of these markers was measured using receiver operating characteristic analysis. RESULTS: Vascular endothelial growth factor (VEGF), interleukin-1α (IL-1α), IL-6, and chemokines including CCL2, CCL5, CXCL1, CXCL8, and CXCL10 were significantly increased in HIC (n = 30) and NHIC (n = 30) patients compared with OAB (n = 28) patients. The significant increases in CXCL8 and CXCL10 were also found in HIC patients compared with NHIC patients. However, there were no significant differences in the other urine markers among the groups. Area under the curves for VEGF, CXCL10, CXCL8, IL-1α, CCL5, CCL2, IL-6, and CXCL1 to detect IC in these patients were 0.87, 0.86, 0.81, 0.80, 0.80, 0.71, 0.66, and 0.50 respectively. CONCLUSIONS: The increases in angiogenesis-associated proteins such as VEGF and CXCL10 may be pathophysiologically important for the development of IC.

Placental Growth Factor in Bladder Cancer Compared to the Diagnostic Accuracy and Prognostic Performance of Vascular Endothelial Growth Factor A.

BACKGROUND/AIM: To evaluate the diagnostic accuracy and prognostic performance of urinary and plasma levels of placental growth factor (PLGF) and provide their comparison with the results of vascular endothelial growth factor A (VEGF-A) in patients with primary and recurrent urinary bladder cancer. MATERIALS AND METHODS: The enzyme-linked immunosorbent assay (ELISA) was used to assess urinary and plasma concentrations of PLGF and VEGF-A in 240 individuals. RESULTS: PLGF levels in urine and plasma were significantly higher in patients with primary bladder cancer than in healthy individuals (p=0.003, p=0.005, respectively). Area under the curve (AUC) of urinary PLGF was 0.68; AUC of plasma PLGF levels was 0.65. Patients with the urine levels of PLGF higher than 82.33 pg/ml had three times higher risk of recurrence. In patients with recurrent bladder cancer, the urinary concentrations of PLGF did not significantly differ from the concentrations in patients without current disease (p=0.61). However, plasma PLGF levels were significantly higher in patients diagnosed with tumor recurrence (p=0.001); AUC of plasma PLGF levels was 0.69. Moreover, patients with plasma levels higher than 10.09 pg/ml had a five-times higher risk of future tumor recurrence. The diagnostic accuracy of PLGF was comparable with VEGF-A. CONCLUSION: From a clinical point of view, PLGF could be considered a valid diagnostic test for the detection of primary and recurrent bladder cancer. In patients with recurrent bladder cancer, plasma PLGF levels can differentiate individuals at risk of tumor recurrence.

Urinary and circulating levels of the anti-angiogenic isoform of vascular endothelial growth factor-A in patients with chronic kidney disease.

INTRODUCTION: The protective effects of vascular endothelial growth factor (VEGF)-A165b on kidney tissue have been suggested in animal studies. However, the relevance of urinary and circulating VEGF-A165b levels in chronic kidney disease patients remains unclear. Therefore, the present study aimed to investigate the urinary and circulating VEGF-A165b levels in patients with chronic kidney disease. METHODS: This observational study enrolled a total of 92 Japanese patients with chronic kidney disease, who had undergone inulin renal clearance measurements for the accurate assessment of measured GFR. Urinary or circulating total VEGF-A and VEGF-A165b levels were measured using enzyme-linked immunosorbent assay. RESULTS: Urinary VEGF-A165b levels were significantly lower in G3a, G3b, and G4+G5 category patients than in G1+G2 category patients. Correlation analysis found that serum creatinine levels, serum cystatin C levels, eGFRcre, eGFRcys, and mGFR were weakly but significantly correlated with urinary VEGF-A165b levels. Additionally, circulating VEGF-A165b levels were significantly higher in G4+G5 category patients than in G1+G2 category patients. CONCLUSION: A low urinary VEGF-A165b level reflects renal dysfunction in the chronic kidney disease stage, while a high circulating VEGF-A165b level cannot be attributed to decreased renal clearance.

The predictive value of urinary vascular endothelial growth factor (VEGF) on worsening kidney function in proteinuric chronic kidney disease.

OBJECTIVES: To evaluate the role of urinary vascular endothelial growth factor (VEGF) as an early predictor of chronic kidney disease (CKD) progression in patients with glomerular diseases. METHODS: We prospectively included patients with proteinuria and CKD grade 1 - 5 due to glomerular disease at the time of kidney biopsy. At baseline, we collected demographics, comorbidities, smoking history, serum creatinine (sCr), proteinuria, and urinary VEGF in collected 24-hour urine. The primary outcome was a 50% increase in sCr at last follow-up. Binary regression was used to explore the impact of urinary biomarkers adjusted for baseline patient characteristics on the outcome. RESULTS: From July 2011 to September 2012 we included 49 patients aged 45.2 ± 14.8 years, 43% female, with different glomerular diseases. We followed them for 29 ± 11 months. Twelve out of 49 (22%) patients met the primary outcome. The patients with a 50% increase in sCr at last follow-up had a significantly higher baseline sCr (193 ± 101 vs. 127 ± 84; p = 0.014) and higher urinary VEGF/creatinine in 24-hour urine (7.7 ± 6.4 vs. 3.0 ± 4.0; p = 0.005). When we added both sCr and urinary VEGF/creatinine to the binary regression model, the correlation with baseline sCr was not significant (OR 1.01; 95% CI 1.00 - 1.01; p = 0.184), while urinary VEGF/creatinine remained significant (OR 1.18; 95% CI 1.04 - 1.35; p = 0.008). Baseline patient characteristics, such as age, gender, body mass index, sCr, proteinuria, smoking status, histopathologic diagnosis, concomitant arterial hypertension, and time to last follow-up did not influence the primary outcome. CONCLUSIONS: The urinary VEGF/creatinine ratio in 24-hour urine seems to independently predict worsening of chronic kidney disease in patients with glomerular diseases.
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