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The acrosome located within the mammalian sperm head is essential for successful fertilization, as it enables the sperm to penetrate the extracellular layers of the oocyte and fuse with oolemma. However, the mammalian acrosomal vesicle is no longer considered to contain only hydrolytic enzymes. Using label-free nano-scale liquid chromatography tandem mass spectrometry (nLC-MS/MS) proteomics, we identified a total of 885 proteins in the acrosome isolated from spermatozoa obtained from cauda epididymis of free-living house mice Mus musculus musculus contains a total of 885 proteins. Among these, 334 proteins were significantly enriched in the acrosome thus representing 27.3% of the whole proteome of the intact sperm. Importantly, we have detected a total of nine calycins while eight of them belong to the lipocalin protein family. In mice, lipocalins are involved in multi-level chemical communication between individuals including pheromone transport and odor perception. Using an indirect immunofluorescence assay, we demonstrated that lipocalin 5 (LCN5) is expressed in the mouse germ cells, and after completing spermatogenesis, it remains localized in the sperm acrosome until the last step of the extratesticular maturation, the acrosome reaction. The presence of lipocalins in the acrosome and acrosome-reacted sperm suggests their original role as chelators of organic and potentially toxic compounds resulting from ongoing spermiogenesis. Along with this evidence, detected mitochondrial (e.g., a subunit of the cytochrome c oxidase MTCO1) and proteasomal proteins (subunits of both 20 S core proteasome [PSMA2, PSMBs] and 19 S regulatory particle [PSMDs]) in acrosomes provide further evidence that acrosomes could also function as `waste baskets` after testicular sperm maturation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The prevalence of chronic kidney disease (CKD) is increasing worldwide, and the mortality rate continues to be unacceptably high. The biomarkers currently used in clinical practice are considered relevant when there is already significant renal impairment compromising the early use of potentially successful therapeutic interventions. More sensitive and specific biomarkers to detect CKD earlier on and improve patients' prognoses are an important unmet medical need. The aim of this review is to summarize the recent literature on new promising early CKD biomarkers of renal function, tubular lesions, endothelial dysfunction and inflammation, and on the auspicious findings from metabolomic studies in this field. Most of the studied biomarkers require further validation in large studies and in a broad range of populations in order to be implemented into routine CKD management. A panel of biomarkers, including earlier biomarkers of renal damage, seems to be a reasonable approach to be applied in clinical practice to allow earlier diagnosis and better disease characterization based on the underlying etiologic process.

Beta-trace protein (BTP), a low molecular weight protein of 23-29 kDa, has been proposed as a promising biomarker to estimate residual renal function (RRF) in patients on maintenance hemodialysis (HD). Indeed, BTP is cleared by native kidney but not during conventional HD session. By contrast, the removal rate of BTP using convective processes (mainly hemodiafiltration [HDF]) and peritoneal dialysis (PD) has been little or not investigated. Therefore, an aim of this study was to evaluate the impact of dialysis procedures (high-flux HD, on-line post-dilution HDF and PD) on BTP removal in comparison with beta-2 microglobulin (B2M) and cystatin C (CYSC) removals after a single session. In addition, the ability of BTP to predict RRF in PD was assessed. This observational cross-sectional study included a total of 82 stable chronic kidney disease patients, 53 patients were on maintenance dialysis (with n = 26 in HD and n = 27 in HDF) and 29 were on PD. Serum concentrations of BTP, B2M, and CYSC were measured (a) before and after a single dialysis session in HD and HDF anuric patients to calculate reduction percentages, (b) in serum, 24-hour-dialysate and 24-hour-urine in PD patients to compute total, peritoneal, and urinary clearance. RRF was estimated using four equations developed for dialysis patients without urine collection and compared to the mean of the urea and creatinine clearances in PD. The concentrations of the three studied molecules were significantly reduced (P < .001) after dialysis session with significantly higher reduction ratio using HDF compared to HD modality (P < .001): BTP 49.3% vs 17.5%; B2M 82.3% vs 69.7%; CYSC 77.4% vs 66% in HDF and HD, respectively. In non-anuric PD patients, B2M and CYSC were partly removed by peritoneal clearance (72.3% and 57.6% for B2M and CYSC, respectively). By contrast, BTP removal by the peritoneum was negligible and a low bias for the BTP-based equation to estimate RRF (-1.4 mL/min/1.73 m 2 ) was calculated. BTP is significantly removed by high-flux HD or HDF, thereby compromising its use to estimate RRF. By contrast, BTP appears as a promising biomarker to estimate RRF in PD patients since it is not affected by peritoneal clearance, unlike B2M and CYSC, and it is well correlated to RRF., (© 2020 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.)

Cerebrospinal fluid (CSF) leaks can occur when there is communication between the intracranial cavities and the external environment. They are a common and serious complication of numerous procedures in otolaryngology, and if not treated, persistent leaks can increase a patient's risk of developing life-threatening complications such as meningitis. As it is not uncommon for patients to exhibit increased secretions postoperatively, distinguishing normal secretions from those containing CSF can be difficult. Currently, there are no proven, available tests that allow a medical provider concerned about a CSF leak to inexpensively, rapidly, and noninvasively rule out the presence of a leak. The gold standard laboratory-based test requires that a sample be sent to a tertiary site for analysis, where days to weeks may pass before results return. To address this, our group recently developed a semiquantitative, barcode-style lateral-flow immunoassay (LFA) for the quantification of the beta-trace protein, which has been reported to be an indicator of the presence of CSF leaks. In the work presented here, we created a rapid diagnostic test kit composed of our LFA, a collection swab, dilution buffers, disposable pipettes, and instructions. Validation studies demonstrated excellent predictive capabilities of this kit in distinguishing between clinical specimens containing CSF and those that did not. Our diagnostic kit for CSF leak detection can be operated by an untrained user, does not require any external equipment, and can be performed in approximately 20 min, making it well suited for use at the point of care. This kit has the potential to transform patient outcomes.

Objective: A cerebrospinal fluid leak is one of the most serious complications in otolaryngology. It may occur as a result of injury to the skull base, typically traumatic or iatrogenic. While the presence of a leak is often discerned in the emergent setting, distinguishing normal secretions from those containing cerebrospinal fluid can be difficult during postoperative visits in the clinic. As most current laboratory-based assays are labor intensive and require several days to result, we aim to develop a more user-friendly and rapid point-of-care cerebrospinal fluid detection device., Study Design: Our laboratory developed a barcode-style lateral-flow immunoassay utilizing antibodies for beta-trace protein, a protein abundant in and specific for cerebrospinal fluid, with a concentration of 1.3 mg/L delineating a positive result., Setting: Tertiary medical center., Subjects and Methods: Tests with known concentrations of resuspended beta-trace protein and the contents of discarded lumbar drains (presumed to contain cerebrospinal fluid) were performed to validate our novel device., Results: Our results demonstrate the ability of our device to semiquantitatively identify concentrations of beta-trace protein from 0.3-90 mg/L, which is within the required range to diagnose a leak, thus making beta-trace protein an excellent target for rapid clinical detection., Conclusion: Herein we detail the creation and initial validation of the first point-of-care cerebrospinal fluid detection device. This device is a feasible method to more efficiently and cost-effectively identify cerebrospinal fluid leaks, minimize costs, and improve patient outcomes.

Recently, studies on human salt taste sensitivity demonstrated that sodium chloride (NaCl) sensitive and non-sensitive subjects differed in their salivary proteome and, in particular, in endopeptidase activity. In order to investigate individual's NaCl sensitivity and the role of endoprotease activity in salt taste perception, 20 panellists were classified according to NaCl sensitivity and saliva samples collected. A targeted protein quantitation by means of selected-reaction-monitoring (SRM) mass spectrometry and stable-isotope incorporation revealed the joint abundance of lysozyme C and lipocalin-1 to be indicative for non-sensitive subjects. Sensory studies performed after oral challenge with the serine-type endopeptidase trypsin demonstrated a salt enhancing effect which was assumed to be due to an in-vivo generation of salt-modulating peptides as shown by LC-SWATH-MS. Amongst those, the tetrapeptide PLWR was found to elicit salty taste enhancing activity above an extraordinarily low taste threshold concentration of 6.5 μmol/L., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

A study on gender differences in the normal range of biomarkers in porcine saliva samples has the scope for further attention. In the present study, the salivary protein profiles of age-matched healthy male and female finishing pigs were compared. The levels of salivary adenosine deaminase (ADA) activity, haptoglobin (Hp) and C-reactive protein (CRP) have been quantified in 32 male and 32 female pigs to ensure the presence of gender effect on the median levels of salivary biomarkers. Moreover, the total salivary protein content was quantified and compared. The overall salivary protein distribution was compared with SDS-PAGE in 14 male and 14 female pigs and the possible gender influence in the salivary protein profile was analysed by 2DE in 6 male and 6 female pigs. Statistically significant differences were observed in the median values of Hp, CRP, and ADA between male and female pigs (p<0.005). Although the total salivary protein content was not different between the sexes, the salivary protein distribution and profile showed specific gender differences in three proteins of the lipocalin family: the odorant-binding protein, salivary lipocalin and lipocalin 1. These proteins have been related to animal immune status and should be further explored as possible porcine salivary biomarkers., Significance: The biological relevance of the reported research is based on the possible gender influence on the discovery of salivary biomarkers in porcine production. As differences have been reported in the salivary protein distribution in male pigs in comparison to that of female pigs, the normal-range values, according to gender, of the newly discovered biomarkers should be explored and defined prior to its application in the porcine production system. A hormonal sexual influence is highly hypothesized., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Female house mice produce pheromone-carrying major urinary proteins (MUPs) in a cycling manner, thus reaching the maximum urinary production just before ovulation. This is thought to occur to advertise the time of ovulation via deposited urine marks. This study aimed to characterize the protein content from the house mouse vaginal flushes to detect putative vaginal-advertising molecules for a direct identification of reproductive states. Here we show that the mouse vaginal discharge contains lipocalins including those from the odorant binding (OBP) and major urinary (MUP) protein families. OBPs were highly expressed but only slightly varied throughout the cycle, whilst several MUPs were differentially abundant. MUP20 or 'darcin', was thought to be expressed only by males. However, in females it was significantly up-regulated during estrus similarly as the recently duplicated central/group-B MUPs (sMUP17 and highly expressed sMUP9), which in the mouse urine are male biased. MUPs rise between proestrus and estrus, remain steady throughout metestrus, and are co-expressed with antimicrobial proteins. Thus, we suggest that MUPs and potentially also OBPs are important components of female vaginal advertising of the house mouse.

Background: Cerebrospinal fluid (CSF) leakage is a rare condition that can potentially lead to the development of serious complications. In the last decade, β-trace protein (β-TP) has been shown to be a valuable immunological biomarker that allows prompt and non-invasive identification of CSF leakage. At our institution, the measurement of β-TP has been included in the diagnostic work-up of CSF leakage for more than 10 years. According to our diagnostic algorithm, the presence of CSF in secretion is excluded when β-TP values are <0.7 mg/L, whereas β-TP values ≥1.3 mg/L indicate the presence of CSF in secretion. β-TP values between 0.7 and 1.29 mg/L indicate the presence of CSF if the β-TP ratio (β-TP secretion/β-TP serum) is ≥2. This study aimed to validate this diagnostic algorithm using clinically defined nasal/ear secretions., Methods: We performed a retrospective statistical analysis of three β-TP interpretation strategies using data of 236 samples originating from 121 patients with suspect CSF leakage received at our laboratory between 2004 and 2012., Results: The highest odds ratio was obtained when the proposed algorithm has been used for the interpretation of β-TP results, showing a sensitivity of 98.3% and a specificity of 96%. Positive and negative predictive values were 89.2% and 99.4%, respectively., Conclusions: Our data suggest that the proposed β-TP interpretation algorithm is a valuable tool for the diagnosis of CSF leakage in the clinical practice.

Corneal neovascularization (CNV) was induced in Balb/c mice by alkali burns in the central area of the cornea with a diameter of 2.5mm. After fourteen days, the cornea from one eye was collected for histological staining for CNV examination, while the cornea from the other eye of the same mouse was harvested for proteomic analysis. The label-free quantitative proteomic approach was applied to analyze five normal corneal tissues (normal group mice n=5) and five corresponding neovascularized corneal tissues (model group mice n=5). A total of 2124 proteins were identified, and 1682 proteins were quantified from these corneal tissues. Among these quantified proteins, 290 proteins were significantly changed between normal and alkali burned corneal tissues. Of these significantly changed proteins, 35 were reported or predicted as angiogenesis-related proteins. Then, these 35 proteins were analyzed using Ingenuity Pathway Analysis Software, resulting in 26 proteins enriched and connected to each other in the protein-protein interaction network, such as Lcn-2, αB-crystallin and Serpinf1 (PEDF). These three significantly changed proteins were selected for further Western blotting validation. Consistent with the quantitative proteomic results, Western blotting showed that Lcn-2 and αB-crystallin were significantly up-regulated in CNV model, while PEDF was down-regulated. This study provided increased understanding of angiogenesis-related proteins involved in corneal vascular development, which will be useful in the ophthalmic clinic of specifically target angiogenesis., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Group B streptococcus (GBS) infection in pregnancy is a major cause of maternal and neonatal morbidity. An understanding of the mechanisms responsible for GBS persistence in the genital tract, as well as recognition of host defenses employed to combat its presence, are crucial to our efforts to reduce maternal GBS colonization and prevent the acquisition of neonatal infections. However, alterations in vaginal immunity in response to GBS colonization in pregnant women remain incompletely defined. Whether GBS modulates autophagy, a major host defense mechanism and contributor to the control of intracellular microbial infections, also remains unclear., Objective: We sought to identify differences in the extent of autophagy as well as in the concentration of biomarkers previously shown to be involved in vaginal innate immunity between GBS-positive and GBS-negative pregnant women., Study Design: We performed a prospective cohort study of healthy pregnant women, who had vaginal secretions obtained at 35-37 weeks of gestation, just prior to the standard GBS rectovaginal sample collection. The contents of the swabs were released into tubes containing 1 mL of sterile phosphate-buffered saline. Samples were centrifuged, and supernatant and cell pellet fractions were collected and stored separately at -80°C until used for analysis. Epithelial cells were then lysed, and the extent of autophagy was determined by measuring the residual level of p62 remaining in the cytoplasm. p62 is a protein that is consumed during autophagy, and so its concentration detectable in the cytoplasm is inversely related to the extent of autophagy induction. The intracellular level of the inducible 70-kDa heat shock protein (hsp70), an inhibitor of autophagy, was also measured. The cell-free fraction was assayed for D- and L-lactic acid, neutrophil gelatinase-associated lipocalin, extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinase (MMP)-8, alpha amylase, hyaluronan, and total protein. Laboratory personnel were blinded to all clinical data., Results: There were 145 women included in the study, of which 45 (31%) were culture-positive for GBS. Vaginal cells from GBS-positive women had elevated intracellular levels of p62 (2.1 vs 0.7 pg/mL, P < .01) and hsp70 (16.9 vs 9.6 ng/mL, P = .03) as compared to GBS-negative women. The p62 and hsp70 levels were highly correlated in both groups of subjects (P < .01). In vaginal fluid, concentrations of neutrophil gelatinase-associated lipocalin (1.1 vs 0.7 ng/μg total protein, P = .01), MMP-8 (21.9 vs 11.1 pg/μg total protein, P = .01), and extracellular MMP inducer (8.8 vs 7.2 pg/μg total protein, P = .03) were highest in GBS-positive women. There were no differences in the concentrations of D- and L-lactic acid, alpha amylase, or hyaluronan between the 2 groups of women., Conclusion: The inhibition of autophagy in vaginal epithelial cells by GBS-induced hsp70 production is associated with its persistence. Concurrently, alterations in components known to influence vaginal bacterial colonization or facilitate microbial passage to the upper genital tract also occur in relation to GBS carriage., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Oestrous suppression by intrauterine devices (IUDs) is caused by prolongation of luteal function, but the biological mechanism is unknown. The aim of the study was to investigate mechanisms which could explain the action of IUDs. Thirty mares were age-matched and either inseminated (AI, n = 15) or fitted with an IUD (IUD, n = 15) and subsequently divided into four groups: AI-P, pregnant (n = 8); AI-N, non-pregnant (n = 7); IUD-P, prolonged luteal phase (n = 7); and IUD-N, normal luteal phase (n = 8). The median ages were 5.5 and 7 years in AI-P and IUD-P groups and 14 and 11 years in AI-N and IUD-N groups, respectively. On Day 15 after ovulation, an endometrial biopsy was obtained to study histomorphological and immunohistochemical expression patterns of uterine proteins (uteroferrin, UF; uterocalin, UC; uteroglobin, UG), oestrogen and progesterone receptors (ER, PR), proliferation marker Ki-67 and content of inflammatory cells. Expression of UF was higher in IUD mares; the difference between pregnant and IUD-P mares was significant. Mares exhibiting a prolonged luteal phase (AI-P, IUD-P) showed only mild angiosclerosis and lower expression of both ER and PR than mares with a normal luteal phase (AI-N, IUD-N). No significant differences were detected in the numbers of inflammatory cells, with the exception of macrophages, which were more numerous in AI-P than AI-N mares. Although inflammatory cells were not detected in IUD mares, increased UF levels may indicate chronic inflammation. Young age and normality of the endometrial blood vessels may improve the efficacy of IUDs., (© 2015 Blackwell Verlag GmbH.)

Background: Due to the lack of nephrotoxic activity, proliferation signal inhibitors (PSI) such as everolimus are recommended for immunosuppression after heart transplantation, but the assessment of renal function in patients receiving PSI has led to conflicting results. We examined renal integrity and function using neutrophil gelatinase-associated lipocalin (NGAL) and conventional markers [plasma creatinine, cystatin C, urine albumin, α1-microglobulin (α1M)] in heart transplant patients, who underwent conversion to everolimus due to allograft vasculopathy, graft rejection episodes, or renal function deterioration, and in patients maintained on calcineurin inhibitors (CNI)., Methods: This cross-sectional study included 121 consecutive heart transplant recipients: 44 patients received CNI-free immunosuppressive therapy with everolimus and 77 patients received CNI. Renal parameters were determined in plasma and urine samples using standard enzymatic or immunochemical methods., Results: Heart transplant recipients receiving everolimus therapy had significantly lower NGAL concentrations in plasma [median (95% CI): 128 (97-176)ng/mL vs. 252 (224-283)ng/mL, p<0.001] and urine [median (95% CI): 6.4 (4.5-7.6)ng/g vs. 15.7 (10.2-25.9)ng/g creatinine, p<0.001]. In contrast, no significant differences were observed between everolimus- and CNI-treated groups with regard to creatinine and cystatin C, as well as urine albumin and α1M levels. Significant correlations were noted between plasma NGAL and creatinine (r=0.42, p<0.001), cystatin C (r=0.44, p<0.001), N-terminal brain natriuretic propeptide (r=0.31, p<0.01) and indicators of chronic inflammation [lipoprotein-associated phospholipase A2 (Lp-PLA2), r=0.31, p<0.01] and soluble CD40 ligand (sCD40L, r=0.22, p<0.05), and between urinary NGAL and α1M (r=0.21, p<0.05). Multiple regression analysis indicated that cystatin C and Lp-PLA2 were the best predictors of plasma NGAL., Conclusion: The present study documents reduced plasma and urinary NGAL levels in the absence of differences in conventional renal parameters in patients on CNI-free immunosuppressive therapy with everolimus. These results support favorable effects of everolimus on renal integrity in heart transplant recipients., (Copyright © 2015 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.)

Purpose: Neutrophil gelatinase associated lipocalin, also known as lipocalin-2, is a 25 kDa protein now considered the biochemical gold standard for the early diagnosis of acute kidney injury. Recently lipocalin-2 was suggested to have an important role in several human neoplasias. In this study we assess lipocalin-2 expression in 2 renal tumor types and analyze its association with clinicopathological parameters and prognosis., Materials and Methods: The study group included 189 patients who underwent surgery for renal lesions between 2003 and 2013. Of these patients 105 had clear cell renal cell carcinoma and 84 had papillary renal cell carcinoma. The association of lipocalin-2 immunoexpression and clinicopathological characteristics was evaluated. Cox proportional hazard regression was performed to estimate the prognostic significance of potential confounders in predicting overall and disease-free survival., Results: Lipocalin-2 expression in different histotypes of analyzed tumors was highly and significantly associated with papillary renal cell carcinoma (p <0.001). In papillary renal cell carcinoma high lipocalin-2 expression was associated with high Fuhrman grade (p <0.001), tumor size greater than 7 cm (p = 0.007), increased TNM stage (p <0.001) and lymph node metastasis (p = 0.009). On univariable and multivariable Cox survival analyses of papillary renal cell carcinoma, after a median followup of 49.1 months (range 7 to 136) lipocalin-2 expression was associated with reduced overall survival (HR 4.10, 95% CI 1.19-14.14, p = 0.026) and disease-free survival (HR 2.56, 95% CI 1.01-6.48, p = 0.047)., Conclusions: Lipocalin-2 over expression may be a prognostic factor in decreased overall and disease-free survival in papillary renal cell carcinoma. The association of lipocalin-2 over expression and poor prognosis with papillary renal cell carcinoma may have potential diagnostic and therapeutic utility., (Copyright © 2015 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objective: Neutrophil gelatinase-associated lipocalin (NGAL) is a component of innate immunity that prevents iron uptake by microorganisms. We evaluated whether NGAL was present in vaginal fluid and whether concentrations were altered in women with bacterial vaginosis (BV) or vulvovaginal candidiasis (VVC)., Methods: Vaginal secretions from 52 women with VVC, 43 with BV, and 77 healthy controls were assayed by enzyme-linked immunosorbent assay for NGAL and for concentrations of L-lactic acid., Results: The median concentration of NGAL in vaginal fluid was significantly higher in control women (561 pg/mL) than in women with BV (402 pg/mL; P = .0116) and lower in women with VVC (741 pg/mL; P = .0017). Median lactic acid levels were similar in controls (0.11 mmol/L) and women with VVC (0.13 mmol/L) and were lower in women with BV (0.02 mmol/L; P < .0001). The NGAL and lactic acid concentrations were highly correlated (P < .0001)., Conclusion: A decrease in Lactobacilli and/or lactic acid plus the absence of leukocytes results in lower vaginal NGAL levels that might facilitate the growth of bacteria associated with BV., (© The Author(s) 2015.)

Sepsis is a major healthcare problem and a leading cause of death worldwide. There is no dependable diagnosis, and treatment for this condition remains mainly supportive. The etiology of sepsis is related to an overwhelming inflammatory response. In this regard, the antimicrobial protein lipocalin-2 (Lcn2) has been associated with several inflammatory conditions, but its contribution to polymicrobial sepsis is unclear. Polymicrobial sepsis was induced by cecal ligation and puncture (CLP), and Lcn2 mRNA levels and protein expression were measured in liver and lung tissues. We observed that Lcn2 expression was robustly induced in liver and lung of C57BL/6 J (B6) mice, and remained elevated during the stage of innate immune dysfunction observed in sepsis. This response was different in A/J mice, suggesting a contribution of the genetic background, probably due to differences in IL-10 expression between these two mouse strains. Indeed, IL-10 was found to regulate Lcn2 expression in both primary and J774A.1 macrophages. Thus, Lcn2 expression is highly regulated during CLP-induced sepsis, suggesting that this antimicrobial protein could have a role as a potential biomarker for the diagnosis of sepsis., (© The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.)

Objective: Neutrophil gelatinase-associated lipocalin (NGAL) is produced in response to tubular injury. Contrast-induced acute kidney injury (CI-AKI) is associated with adverse outcomes in chronic kidney disease (CKD) patients. We sought to characterize blood NGAL level and the degree of kidney injury in CKD patients who underwent coronary angiography., Methods: This study was a prospective, blinded assessment of blood samples obtained from patients with estimated glomerular filtration rates (eGFRs) between 15 and 90 mL/min/1.73 m2 undergoing elective coronary angiography with iodinated contrast. Blood NGAL and serum creatinine were measured at baseline, 1, 2, 4, 6, 12, 24 and 48 h after contrast administration., Results: A total of 63 subjects with a mean eGFR of 48.17±16.45 mL/min/1.73 m2 were enrolled. There was a graded increase in baseline NGAL levels across worsening stages of CKD (p=0.0001). Post-procedure NGAL increased from baseline in each stage of CKD. Eight (12.7%) patients were diagnosed with CI-AKI by diagnostic criteria of 2012 KDIGO definition of CI-AKI, and seven (11.1%) patients developed subclinical CI-AKI defined by a twofold or greater rise in NGAL. There was no relationship between baseline eGFR and diabetes on the composite outcome of subclinical and clinical CI-AKI., Conclusions: Baseline and post-procedure NGAL are progressively elevated according to the baseline stage of CKD. Using a twofold rise in NGAL, 46.7% of composite CI-AKI is detected and complements the 53.3% of cases identified using KDIGO criteria. Traditional risk predictors were not independently associated with this composite outcome.

Background: We describe a case of a 56-year-old woman who developed a recurrent pleural effusion after a thoracoscopic resection of an anterior bulging thoracic disc hernia (level D9-D10). Despite several evacuating pleural punctions, dyspnea reoccurred due to recurrent pleural effusion, the same side as the disc resection. Because of increasing headache after each punction, a subarachnoidal pleural fistula (SAPF) was suspected. Although magnetic resonance imaging (MRI) showed features suggestive of SAPF, there was not enough evidence to justify a new thorascopy., Methods: Cerebrospinal fluid (CSF) leakage into the thoracic and abdominal cavity has been described as a result of trauma or surgery. Detection of beta-trace protein (BTP, a brain-specific protein) has been described to detect CSF fistulae causing rhino- and otoliquorrhea. Similarly, BTP determination could be used to identify the presence of CSF at other anatomical sites such as the thoracic cavity. Therefore, we decided to determine the concentration of BTP in the pleural effusion of this patient. BTP was assayed using immunonephelometry., Results: The patient's BTP pleural fluid concentration was 14·0 mg/l, which was a 25-fold increase compared with the BTP serum concentration. After insertion of a subarachnoidal lumbal catheter, a video-assisted thorascopy was performed. Leakage of liquor through the parietal pleura into the thoracic cavity was observed. The SAPF was closed using a durasis patch and DuraSeal®. Postoperatively, there was no reoccurrence of pleural fluid., Conclusions: SAPF has to be included to the differential diagnosis of patients with persistent pleural effusion after spinal surgery. This case illustrates the importance of BTP in diagnosing SAPF, especially in cases where major therapeutic consequences may need to be drawn.

Bacterial vaginosis is a common reproductive infection in which commensal vaginal lactobacilli are displaced by a mixed population of pathogenic bacteria. Bacterial vaginosis increases susceptibility to HIV, and it has been suggested that host innate immune responses to pathogenic bacteria contribute to enhanced infection, yet the cellular mechanisms mediating the increased HIV susceptibility remain uncharacterized. We evaluated the HIV-enhancing effects of bacterial vaginosis by inoculating endocervical epithelia with Atopobium vaginae, a bacterial vaginosis-associated bacteria, and assaying secreted factors for HIV-enhancing activity. When epithelia and A. vaginae were cocultured, we observed increased HIV-enhancing activity mediated by secreted low molecular weight factors. From this complex mixture we identified several upregulated host proteins, which functioned in combination to enhance HIV infection. These studies suggest that the host immune response to bacterial vaginosis-associated bacteria results in the release of HIV-enhancing factors. The combined activity of bacterial vaginosis-induced proteins likely mediates HIV enhancement.

Neutrophil gelatinase-associated lipocalin (NGAL) is a candidate diagnostic biomarker for acute kidney injury (AKI). Since there is no specific treatment to reverse AKI, a good biomarker such as NGAL can increase the performance of clinical care. Therefore, a timely, specific and sensitive assay for detecting NGAL is critical for clinical determination. In this study, we established a solid-phase proximity ligation assay for the detection of NGAL using polyclonal antibodies conjugated with a pair of oligonucleotides. The data are read out as the Ct value via quantitative real-time polymerase chain reaction (qPCR). Our results demonstrate that this new assay performs with good specificity and sensitivity for detection of NGAL spiked in buffer or serum, which indicates that the solid-phase proximity ligation technique is a promising tool for diagnostics in clinical decisions.

β-Trace protein (BTP), also known as lipocalin prostaglandin D2 synthase (L-PGDS; encoded by the PTGDS gene), is a low-molecular-weight glycoprotein and an emerging novel marker of glomerular filtration rate. BTP is an important constituent of cerebral spinal fluid and is found in much lower concentrations in blood. Its serum origin and renal handling remain poorly understood. Unlike serum creatinine, BTP is not physiologically inert. It possesses both ligand-binding and enzymatic properties. BTP catalyzes the conversion of prostaglandin H2 (PGH2) to PGD2. PGD2 is an eicosanoid involved in a variety of important physiologic processes, including platelet aggregation, vasodilation, inflammation, adipogenesis, and bone remodeling. Several studies now have documented BTP's strong association with glomerular filtration rate, end-stage renal disease, cardiovascular disease, and death in a variety of different patient populations. This review provides an overview of the biochemistry, physiology and metabolism, biological functions, and measurement of BTP; summarizes the evidence for BTP as a marker of both kidney function and cardiovascular disease; and then considers the interplay between its biological properties, serum concentration, and patient outcomes., (Copyright © 2014 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Background: The purpose of this study is to determine whether baseline salivary inflammatory biomarkers could discriminate between different clinical levels of disease and/or detect clinical changes over a 3-week stent-induced biofilm overgrowth (SIBO) period., Methods: A total of 168 participants were enrolled in a 21-day experimental gingivitis investigation and grouped according to clinical measures of periodontal status of health and diseased individuals representing each of five biofilm gingival interface (BGI) periodontal groups: 1) health, all probing depth (PD) <3 mm and bleeding on probing (BOP) <10%; 2) gingivitis, all PD <3 mm and BOP ≥10%; 3) periodontitis (P)1, ≥1 site with PD >3 mm and BOP ≤10%; 4) P2, ≥1 site with PD >3 mm and BOP >10% but ≤50%; and 5) P3, ≥1 site with PD >3 mm and BOP >50%. Stents were used to prevent plaque removal during brushing over one maxillary and one mandibular posterior dental sextant for 21 days. Clinical periodontal parameters and unstimulated saliva were collected at screening, baseline, and each week during SIBO. Saliva samples were assessed for levels of 13 different biomarkers by multiplex immunoassay., Results: Higher salivary levels of interleukin (IL)-1β, matrix metalloproteinase (MMP)-3, MMP-8, MMP-9, and neutrophil gelatinase-associated lipocalin (NGAL) were found in diseased groups compared with the healthy group at baseline. Conversely, higher IL-1 receptor antagonist (ra) levels were found in healthy patients at baseline. In addition, during SIBO, MMP-1, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 levels increased across all participant groups. A stepwise linear regression model using all salivary biomarkers demonstrated that, at baseline, increased IL-1ra (P = 0.004) and IL-6 (P = 0.009) were significantly associated with change in PDs during SIBO., Conclusions: In summary, this investigation supports salivary levels of IL-1ra and IL-6 as potential indicators for PD changes during induced gingival inflammation. In addition, participants from the BGI-P3 group (severe periodontitis) demonstrated elevated baseline levels of IL-1β, MMP-3, MMP-8, MMP-9, and NGAL compared with the other study groups, strengthening the relevance of participants' biologic phenotype on expression of salivary biomarkers.

Aim of the study is to evaluate the expression of CK19, galectin-3, HBME-1, CD56, claudin-1 and neutrophil gelatinase-associated lipocalin (NGAL) in papillary thyroid carcinoma (PTC), including classic, follicular variant and micro-carcinoma. Peritumoral benign thyroid tissues were used as a control (C). Immunohistochemical staining with the EnVision detection system was performed on 59 formalin-fixed, paraffin-embedded thyroid tissues, including 43 PTC and 16 C. CK19, galectin-3, HBME-1, claudin-1 and NGAL were positive in most PTC, but were negative or showed focal weak staining in C. CD56 was positive in C, but absent in PTC. For a single analyzed tumor marker, the sensitivity of the above six thyroid tumor markers was 100%, 95.3%, 86%, 79.1%, 90.7%, 93%, respectively. The specificity was 56.25%, 100%, 100%, 100%, 100%, 100%, respectively. For the combination of the six tumor markers, the sensitivity, specificity, positive predictive value, negative predictive value and the diagnostic accuracy were all 100%, with co-expression of at least four indices. In the diagnosis of PTC, combined application of the above tumor markers is recommended. It can be diagnosed as PTC clearly when thyroid lesions are positive for at least four of the above tumor markers [CD56⁻], and can be excluded as PTC definitely when at least four tumor markers are negative [CD56⁺].

Background: Lipocalin-2 (Lcn-2) is expressed in human neutrophils and epithelial cells, particularly in the presence of inflammation or cancer. It was shown to be highly expressed in various human cancers. Increased protein levels were associated with decreased survival of patients with breast or gastric cancer. The main focus of this work was to analyze the implication of Lcn-2 up-regulation in the genesis of colon cancer., Methods: Expression of Lcn-2 was analyzed in colorectal carcinoma cell lines, paired colorectal carcinoma tissues, and regular mucosa by Western blot analysis. Lcn-2 immunohistochemical staining was performed in 192 colorectal carcinoma resection specimens and correlated with clinicopathologic parameters., Results: Western blot analysis of colorectal carcinoma tissues demonstrated Lcn-2 overexpression in carcinomas as compared with regular mucosa. Immunohistochemical staining revealed Lcn-2 expression in 179 (93.2%) colorectal carcinoma tissues. Intense immunoreactivity was significantly correlated with metastasis (p = 0.042) and UICC stage (p = 0.027). Survival analysis according to the Kaplan-Meier method revealed a significant association between Lcn-2 overexpressing tumors and overall survival (p < 0.001) and disease-free survival (p < 0.001)., Conclusions: Our data provide evidence that Lcn-2 expression is up-regulated with tumor progression and was found to be a predictor of overall survival.

Purpose: To investigate whether allopurinol exerts a protective effect on kidneys by measuring new kidney injury biomarkers (NGALp, NGALu, KIM 1 and IL 18) and analysing the renal function and histology in uninephrectomised rats subjected to ischaemia-reperfusion injury., Methods: Thirty two Wistar rats were randomly allocated to four groups: Sham (S): laparotomy; Control (C): laparotomy and ischaemia-reperfusion in the left kidney; Control Allopurinol (CA): laparotomy and allopurinol at a dose of 100mg·kg 1·d 1; and Allopurinol (A): laparotomy ischaemia-reperfusion in the left kidney and allopurinol at a dose of 100mg·kg 1·d 1. The NGALp, NGALu, KIM 1, IL 18 and creatinine levels and the kidney histology were analysed. The significance level was established as p<0.05., Results: Creatinine level increased in all the groups, with A ≈ C > S ≈ CA. The NGALp, NGALu and IL 18 levels exhibited similar behaviour in all the groups. KIM 1 was higher in group A than C and showed intermediate values in groups S and CA. Severity of injury in the left kidney was greater in groups C and A compared to S and CA., Conclusion: Allopurinol did not exert protective or damaging effects on the kidneys of rats subjected to ischaemia-reperfusion injury.

Background: Mixed venous and arterial ulcers account for approximately 15%-30% of all venous leg ulcerations. Several studies have shown that matrix metalloproteinases (MMPs) and neutrophil gelatinase-associated lipocalin (NGAL) play a central role in the pathophysiology of venous and arterial diseases. Some studies have shown the efficacy of glycosaminoglycans, such as sulodexide (SDX), in treating patients with leg ulcers. The aim of this study was to evaluate clinical effects of SDX and its correlation with MMPs and NGAL expression in patients with mixed arterial and venous leg ulcers., Methods: Patients eligible for this study were of both sexes, older than 20 years, and with a clinical and instrumental diagnosis of mixed ulcer., Results: Fifty-three patients of both sexes were enrolled and divided into two groups by means of randomization tables. Group A (treated group) comprised 18 females and ten males (median age: 68.7 years) treated with standard treatment (compression therapy and surgery) + SDX (600 lipoprotein lipase-releasing units/day intramuscularly) for 15 days followed by SDX 250 lipase-releasing units every 12 hours day orally for 6 months as adjunctive treatment. Group B (control group) comprised 17 females and eight males (median age: 64.2 years) treated with standard treatment only (compression therapy and surgery). The type of surgery was chosen according to anatomical level of vein incompetence: superficial venous open surgery and/or subfascial endoscopic perforating surgery. In all enrolled patients, blood samples were collected in order to evaluate the plasma levels of MMPs and NGAL through enzyme-linked immunosorbent assay. These results were compared to another control group (Group C) of healthy individuals. Moreover, biopsies of ulcers were taken to evaluate the tissue expression of MMPs and NGAL through Western blot analysis. Our results revealed that SDX treatment is able to reduce both plasma levels and tissue expression of MMPs improving the clinical conditions in patients with mixed ulcers., Conclusion: Inhibition of MMPs could represent a possible therapeutic intervention to limit the progression of leg ulceration. In particular, our findings demonstrate the efficacy of SDX in patients with mixed arterial and venous chronic ulcers of the lower limbs.

Objectives: The middle ear cleft connects by membranes to the inner ear and the subarachnoid space particularly in infants. In order to gain more insight about the permeability between the two compartments we quantified the concentration of beta-trace protein - a highly specific marker for CSF and perilymph but not for serum and mucosal effusion - in middle ear secretions from children with otitis media with effusion., Methods: One-hundred and three patients were included and 93 samples from secretory otitis media were collected during myringotomy or explorative tympanotomy. Thirty-eight patients of 103 had to be excluded (36.9%). Of the 93 collected samples from 65 subjects, 82 viscous samples were pre-diluted 1:1 with tyloxapol. In spite of the attempt to pre-dilute the viscous samples, 30 glue-like samples of 93 were not applicable for nephelometry. The final analysis was made on 63 samples of 52 subjects (median age 3 years) which were quantified for beta-trace protein using immunonephelometry., Results: In 3/63 samples the beta-trace protein values were below the detection range (<0.2 mg/L) and in 1/63 it was beyond with 18.3mg/L. The median beta-trace protein value for 59 samples within the measuring range was 2.4 mg/L, range 0.2-14.2., Conclusion: In pediatric middle ear effusions, the beta-trace protein concentration was found to have a high range compared to other body fluids from other studies. In other studies, the values for serum (0.59 mg/L) or mucosal secretion (0.003-0.12 mg/L) were lower and the values in CSF (18.4 mg/L) or perilymphatic fluid (23.5 mg/L) were highest. This finding might indicate a weak barrier between the cerebrospinal fluid space or inner ear fluid compartments on the one side and the tympanic cavity on the other side given the condition of otitis media with effusion. The detection of beta-trace protein might be important to assess the risk of impending complications., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Purpose: To establish the use of selected reaction monitoring (SRM) mass spectrometry for quantification of tear proteins., Methods: Tear samples were collected on multiple occasions (7-10 days) from healthy subjects with contact lens wear (CL = 3) and without contact lens wear (NCL = 4). Tear proteins were denatured using 8M urea, reduced with iodoacetamide, precipitated by acetone, and digested using trypsin. Internal standards were included by adding isotopically-labelled standards of known concentrations to the samples. Lactoferrin, lysozyme, prolactin-induced protein, lipocalin 1, and proline-rich protein 4 were quantified using liquid chromatography-triple quadruple mass spectrometry in conjunction with selected reaction monitoring., Results: The limits of quantification for the selected peptides were below 50 pg/μL. The recovery of peptides from spiked digested tears was greater than or equal to 56% and the coefficient of variation values were less than or equal to 16%. The concentration of lactoferrin (1.20 ± 0.77 μg/μL), lysozyme (2.11 ± 1.50 μg/μL), and lipocalin-1 (1.75 ± 0.99 μg/μL) were consistent with previous ELISA studies. Tear levels of prolactin-induced protein (0.09 ± 0.06 μg/μL) and proline-rich 4 (0.80 ± 0.50 μg/μL) are reported here for the first time., Conclusions: The SRM method can be used for simultaneous detection and quantification of selected proteins in low volumes of human tear samples (2.5 μL per sample) without prior purification of each protein component or need for antibodies.

We have developed a highly sensitive immunoassay using graphene nano platelets (GNPs) for the rapid detection of human lipocalin-2 (LCN2) in plasma, serum, and whole blood. It has the dynamic range, linear range, limit of detection, and analytical sensitivity of 0.6 to 5120, 80 to 2560, 0.7, and 1pg/ml, respectively. It is the most sensitive assay for the detection of LCN2, which has 80-fold higher analytical sensitivity and 3-fold lesser immunoassay duration than the commercially available sandwich enzyme-linked immunosorbent assay (ELISA) kit. The functionalization of microtiter plate (MTP) with GNPs, dispersed in 3-aminopropyltriethoxysilane (APTES), provided the increased surface area that leads to higher immobilization density of capture antibodies. Moreover, the generation of free amino groups on MTP and GNPs by APTES enables the leach-proof covalent crosslinking of anti-human LCN2 capture antibody by its carboxyl groups using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) as the heterobifunctional crosslinker. The anti-LCN2 antibody-bound MTPs were highly stable given that they did not show any significant decrease in their functional activity when stored at 4°C in 0.1M phosphate-buffered saline (PBS) for 8weeks. The developed immunoassay correlated well with the conventional ELISA, thereby demonstrating its high precision and potential utility for highly sensitive analyte detection in industrial and clinical settings., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background/aim: The purpose of this study is to find the frequency of contrast-induced nephropathy (CIN) and to show the risk factors in the development of CIN and the diagnostic utility of serum cystatin C (CysC) and serum and urine neutrophil gelatinase-associated lipocalin (NGAL) during childhood following cardiac angiography., Materials and Methods: In this prospective study, we studied 46 children with congenital heart disease. The levels of serum creatinine, serum CysC, and serum NGAL were measured at 4, 24, and 48 h, while levels of urine NGAL and urine creatinine were measured at 4 to 8 and 48 h following cardiac angiography., Results: According to serum creatinine levels, with a cutoff value of 4.1 mL/kg for development of CIN, sensitivity, specificity, area under the receiver-operating characteristic curve, and positive likelihood ratio were calculated as 69%, 70%, 0.67, and 2.29, respectively. The levels of serum CysC and serum creatinine significantly increased at 4, 24, and 48 h after the application of the contrast agent., Conclusion: The results of this study show that according to the definition of CIN, the incidence of CIN is significantly increased in pediatric patients with congenital heart disease. Moreover, the results support that serum CysC levels may allow the detection of CIN after cardiac angiography, like serum creatinine in present study.

Aim: To evaluate the fecal level of neutrophil gelatinase-associated lipocalin (NGAL) in different behavior of inflammatory bowel disease (IBD)., Materials and Methods: We prospectively included 41 people into the study--30 patients with active IBD and 11 healthy volunteers. The concentration of NGAL in faeces was determined by enzyme immunoassay method., Results: Fecal NGAL level was increased in both UC and CD: in CD--5924.27 ± 2067.6 ng/ml (p < 0.05), in UC--5826.09 ± 891.8 ng/ml (p < 0.05) NGAL levels were higher than in the control group (658.8 ± 237.7 ng/ml). Maximal changes were seen in colonic involvement. NGAL levels increased with the increasing extension of lesion in UC (p < 0.05), while in CD concentration was higher in colitis than in ileitis and ileocolitis (p > 0.05) . NGAL level increased with severity of CD (p < 0.05), in patients with UC difference was not significant. In UC NGAL level was increased with increasing extension of lesions (p < 0.05), in CD this pattern was not marked. Correlation NGAL level with some clinical and laboratory indicators in CD was established. Sensitivity of test in evaluation of exacerbation of IBD was 80%, specificity--90.9%, area under the ROC curve (AUC)--0.9, positive predictive value--96%, negative predictive value--62.5%; positive likelihood ratio--8.8 and negative likelihood ratio--0.22., Conclusions: The fecal concentration of NGAL significantly increased during IBD. With increasing severity and activity of disease level ofNGAL was increased in CD (p < 0.05). Lipocalin-2 values was higher with the increasing extension of lesions in UC (p < 0.05). There has been established the high diagnostic value of the detection of fecal NGAL as a marker of the active phase of IBD.

Fluorescent tagged ligands are commonly used to determine binding to proteins. However, bound and free ligand concentrations are not directly determined. Instead the response in a fluorescent ligand titration experiment is considered to be proportional to the extent of binding and, therefore, the maximum value of binding is scaled to the total protein concentration. Here, a simple model-free method is presented to be performed in two steps. In the first step, normalized bound and free spectra of the ligand are determined. In the second step, these spectra are used to fit composite spectra as the sum of individual components or linear spectral summation. Using linear spectral summation, free and bound 1-Anilinonaphthalene-8-Sulfonic Acid (ANS) fluorescent ligand concentrations are directly calculated to determine ANS binding to tear lipocalin (TL), an archetypical ligand binding protein. Error analysis shows that the parameters that determine bound and free ligand concentrations were recovered with high certainty. The linear spectral summation method is feasible when fluorescence intensity is accompanied by a spectral shift upon protein binding. Computer simulations of the experiments of ANS binding to TL indicate that the method is feasible when the fluorescence spectral shift between bound and free forms of the ligand is just 8 nm. Ligands tagged with environmentally sensitive fluorescent dyes, e.g., dansyl chromophore, are particularly suitable for this method.

The aim of this study was to demonstrate that neutrophil gelatinase-associated lipocalin (NGAL) increased before the onset of microalbuminuria in patients with type 1 diabetes mellitus (T1DM), representing an important biochemical parameter with high sensitivity and specificity to make a precocious diagnosis of "normoalbuminuric" diabetic nephropathy (DN). Serum NGAL (sNGAL) and urinary NGAL (uNGAL) levels were evaluated in a cohort of fifty patients affected by T1DM. They had no signs of clinical nephropathy. Thirty-five healthy subjects (HS) were recruited. sNGAL levels were significantly higher compared with those measured in HS [193.7 (103.2-405.4) vs. 46.4 (39.8-56.2) ng/ml; p < 0.0001], as were uNGAL levels [25.5 (14.2-40.2) vs. 6.5 (2.9-8.5) ng/ml; p < 0.0001]. sNGAL was found to be directly correlated with glycated hemoglobin. uNGAL also positively correlated with albuminuria, whereas an inverse correlation was found with uric acid. After multivariate analysis, significance was maintained for the correlation between uNGAL and microalbuminuria. In ROC analysis, sNGAL showed a good diagnostic profile such as uNGAL. NGAL increases in patients with T1DM, even before diagnosis of microalbuminuria representing an early biomarker of "normoalbuminuric" DN with a good sensitivity and specificity. NGAL measurement could be useful for the evaluation of early renal involvement in the course of diabetes.

Aim: Investigate short-term effects of power brushing following experimental induction of biofilm overgrowth in periodontal disease states., Materials and Methods: Overall, 175 subjects representing each of five biofilm-gingival interface (BGI) periodontal groups were enrolled in a single-blind, randomized study. After stent-induced biofilm overgrowth for 21 days subjects received either a manual or a power toothbrush to use during a 4 weeks resolution phase. At baseline and during induction and resolution, standard clinical parameters were measured. Subclinical parameters included multikine analysis of 13 salivary biomarkers and 16s Human Oral Microbe Identification Microarray (HOMIM) probe analysis of subgingival plaque samples., Results: All groups exhibited significantly greater reductions in bleeding on probing (BOP) (p = 0.002), gingival index (GI) (p = 0.0007), pocket depth (PD) (p = 0.04) and plaque index (p = 0.001) with power brushing compared to manual. When BGI groups were combined to form a shallow PD (PD ≤ 3 mm) and a deep PD group (PD > 4 mm) power brushing reduced BOP and GI in subjects with both pocket depths. Power brushing significantly reduced IL-1β levels at resolution while changes in bacterial levels showed non-significant trends between both brushing modalities., Conclusions: Short-term changes in select clinical parameters and subclinical salivary biomarkers may be useful in assessing efficacy of power brushing interventions in a spectrum of periodontal disease states., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Ultrafiltration failure and peritonitis are the most important limitations of peritoneal dialysis (PD). The aim of our study was to evaluate peritoneum damage through neutrophil gelatinase-associated lipocalin (NGAL), white blood cell (WBC) count and cancer antigen 125 (CA125)., Patients and Methods: Thirty patients with peritonitis and 30 patients undergoing continuous ambulatory peritoneal dialysis (CAPD) were studied for 12 months. In the peritonitis group, blood samples and peritoneal fluid (lp) were collected before the onset of peritonitis, at the onset of peritonitis (T1) and every day until its resolution. CAPD patients were divided into 3 groups according to the treatment received. Long-dwell effluents were collected for NGAL, WBC count and Ca125 assessment., Results: In the peritonitis group, at time T1, NGAL levels were higher compared with baseline values. lpNGAL levels decreased at least 24 hours earlier than peritoneal WBC (lpWBC). At ROC analysis, lpNGAL was characterized by a very good diagnostic profile identifying treatment failure. In CAPD patients, the highest NGAL values were observed in the icodextrin group. An inverse correlation between lpNGAL, pKt/V and peritoneal ultrafiltration volume was also found., Conclusion: Mesothelial cells have an active role in the structural and functional alteration of the peritoneum during PD, and NGAL represents a valid biomarker for peritoneum evaluation.

Supraphysiological O2 concentrations, mechanical ventilation, and inflammation significantly contribute to the development of bronchopulmonary dysplasia (BPD).Exposure of newborn mice to hyperoxia causes inflammation and impaired alveolarization similar to that seen in infants with BPD.Previously, we demonstrated that pulmonary cyclooxygenase-2 (COX-2) protein expression is increased in hyperoxia-exposed newborn mice.The present studies were designed to define the role of COX-2 in newborn hyperoxic lung injury.We tested the hypothesis that attenuation of COX-2 activity would reduce hyperoxia-induced inflammation and improve alveolarization.Newborn C3H/HeN micewere injected daily with vehicle, aspirin (nonselective COX-2 inhibitor), or celecoxib (selective COX-2 inhibitor) for the first 7 days of life.Additional studies utilized wild-type (C57Bl/6, COX-2(+/+)), heterozygous (COX-2(+/-)), and homozygous (COX-2(-/-)) transgenic mice.Micewere exposed to room air (21% O2) or hyperoxia (85% O2) for 14 days.Aspirin-injected and COX-2(-/-) pups had reduced levels of monocyte chemoattractant protein (MCP-1) in bronchoalveolar lavage fluid (BAL).Both aspirin and celecoxib treatment reduced macrophage numbers in the alveolar walls and air spaces.Aspirin and celecoxib treatment attenuated hyperoxia-induced COX activity, including altered levels of prostaglandin (PG)D2 metabolites.Decreased COX activity, however, did not prevent hyperoxia-induced lung developmental deficits.Our data suggest thatincreased COX-2 activity may contribute to proinflammatory responses, including macrophage chemotaxis, during exposure to hyperoxia.Modulation of COX-2 activity may be a useful therapeutic target to limit hyperoxia-induced inflammation in preterm infants at risk of developing BPD., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Periodontal disease is a bacterial infection that destroys the gingiva and surrounding tissues of the oral cavity. Gingival crevicular fluid (GCF) is extracted from the gingival sulcus and pocket. Analysis of biochemical markers in GCF, which predict the progression of periodontal disease, may facilitate disease diagnosis. However, no useful GCF biochemical markers with high sensitivity for detecting periodontal disease have been identified. Thus, the search for biochemical markers of periodontal disease is of continued interest in experimental and clinical periodontal disease research. Using tandem mass tag (TMT) labeling, we analyzed GCF samples from healthy subjects and patients with periodontal disease, and identified a total of 619 GCF proteins based on proteomic analysis. Of these, we focused on two proteins, matrix metalloproteinase (MMP)-9 and neutrophil gelatinase-associated lipocalin (LCN2), which are involved in the progression of periodontal disease. Western blot analysis revealed that the levels of MMP-9 and LCN2 were significantly higher in patients with periodontal disease than in healthy subjects. In addition, ELISA also detected significantly higher levels of LCN2 in patients with periodontal disease than in healthy subjects. Thus, LC-MS/MS analyses of GCF using TMT labeling led to the identification of LCN2, which may be a promising GCF biomarker for the detection of periodontal disease., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Complex interactions existing between cardiac and renal diseases led to define 5 types of so-called cardiorenal syndromes. This classification is based on the organ primarily involved and the acute or chronic failure. The mutual impact of renal and cardiac functions makes it difficult to evaluate and manage patients with cardiorenal syndromes and worsen morbidity and mortality. This review seeks to discuss the place of biomarkers in diagnosis, management and follow-up of patients with cardiorenal syndromes. Biomarkers can be classified as functional (creatinine, cystatin C…) or lesional (neutrophil gelatinase-associated lipocalin, urinary cystatin C…) renal markers and functional (natriuretic peptides…) or lesional (troponin, fatty acid binding protein) cardiac markers. A last kind of biomarkers reflects the dialogue between heart and kidney (renin-angiotensin-aldosteron-system, indicators of activation of arginine vasopressin system) or the systemic impact (inflammation, oxidative stress…). In order to evaluate accurately the complex interactions that are the basis of cardiorenal syndromes, a multi-marker approach seems nowadays necessary.

Renal dysfunction is common in urologic patients, especially in those undergoing nephrectomy for renal cancer. Partial nephrectomy better preserves renal function than radical nephrectomy, but is associated with acute kidney injury related to loss of nephrons and ischemic injury. Ischemic injury may not be reliably assessed using common clinical parameters, such as serum creatinine and urine output, which may delay detection of clinically-significant kidney damage. Molecular markers, such as cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), IL-18 and kidney injury molecule-1 (KIM-1), better quantify the extent of acute ischemic and/or tubular injury than other currently available tools. The use of these and/or other markers may facilitate research to improve outcomes following partial nephrectomy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Aim: The objective of this study was to establish age-dependent urine NGAL (neutrophil gelatinase-associated lipocalin)/creatinine ratio values in healthy children and adolescents., Methods: The study was performed using a random sample of 172 healthy children and adolescents (M-88, F-84), aged median 9.75 (0.2-17.9) years. Urine NGAL concentration was measured using a commercially available ELISA kit (R&D Systems, USA)., Results: Median concentrations of urine NGAL/creatinine in particular age groups were analysed using anova. The differences between the youngest group of children under the age of 6 years and the rest of examined population were statistically significant. There were no differences in urine NGAL/creatinine between other age groups. Statistically significant negative correlation between urine NGAL/creatinine and age of subjects was found (r = -0.29, p < 0.05)., Conclusion: In the study, normative values of urine NGAL/creatinine for subjects aged 0.2-17.9 years have been established. These data may help clinicians and researchers to improve the interpretation of urine NGAL/creatinine ratio in children and adolescents. However, further studies using numerous data should be conducted to add reference values for urine NGAL partitioned by age and gender., (©2013 Foundation Acta Paediatrica. Published by Blackwell Publishing Ltd.)

Acute kidney injury (AKI) is an ominous event in the natural history of cirrhosis. The differential diagnosis of this entity is hampered by the absence of specific biomarkers of tubular damage in cirrhosis. The clinical usefulness of such biomarkers is determined by their effectiveness in the diagnosis of AKI and their ability to provide critical information to ameliorate clinical outcomes and survival. The lack of biomarkers has hindered the development of interventions aimed to improve the prognosis of kidney impairment in cirrhosis. Currently, biomarkers are an area of intense research in nephrology. Emerging genomic and proteomic technologies have revealed novel plasma and urinary biomarkers of AKI. The present article discusses the most promising candidate biomarkers with potential application in cirrhosis, such as NGAL, KIM-1, cystatin-C, IL-18, L-FABP, N-acetyl glucosaminidase and netrin-1, are discussed below., (Copyright © 2013 Elsevier España, S.L. and AEEH y AEG. All rights reserved.)

In this article, we demonstrate how the application of biophysical tools facilitates the design of robust immunoassays. The binding characteristics of the reagents used in an immunoassay determine the assay response to the analyte concentrations. We applied several biophysical methods to obtain pertinent equilibrium and kinetic coefficients and used this information in the design of a microparticle-based immunoassay for detection of neutrophil gelatinase-associated lipocalin (NGAL), which is a new diagnostic marker of acute kidney injury (AKI). We characterized the conformational stability of recombinant human NGAL and the solution phase binding properties of six monoclonal antibodies. A preferred antibody pair was selected on the basis of the affinities of the antibodies and their sandwich pairing capabilities. One of the antibodies was coated on magnetic microparticles, and the second antibody was conjugated with a reporter group. The apparent kinetic rates of the immobilized and conjugated antibodies were measured and used to compute the assay calibration plot for the target concentration range of the analyte at specific incubation times. The experimental assay results were found to be in good agreement with the computed data, confirming that applying biophysical tools provides a solid foundation for immunoassay design and optimization., (Copyright © 2013 Elsevier Inc. All rights reserved.)