Comparison of sVCAM-1 and sICAM-1 levels in maternal serum and vaginal secretion between pregnant women with preterm prelabour ruptures of membranes and healthy pregnant women.

OBJECTIVE: The study aims to evaluate the maternal serum and the vaginal fluid levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecular (sICAM-1) in pregnant women complicated by preterm prelabour ruptures of membranes (PPROM). MATERIALS AND METHODS: The prospective case control study included 34 pregnant women with PPROM and 34 healthy pregnant women. Patients with additional diseases, a smoking habit and vaginal bleeding, as well as those using antibiotics, during the study period were not included in the study. Cervicovaginal fluid and serum samples were taken during the patients' admission. The demographic data, maternal serum and vaginal fluid sVCAM-1 and sICAM-1, C reactive protein (CRP) and leukocyte counts were noted for all pregnant women included in the study. The sVCAM-1 and sICAM-1 levels were measured by enzyme-linked immunosorbent assay kits. RESULTS: In pregnant women with PPROM, the serum leukocyte (mean ± SD =11.41 ± 1.067 versus 9.18 ± 1.56, p < .0001), serum sVCAM-1 (median 771.20 versus 704.60 ng/ml, p < .001), sICAM-1 (mean ± SD 213.10 ± 35.59 ng/ml versus 188.11 ± 37.35 ng/ml, p = .06), vaginal sVCAM-1 (median 208.00 versus 140.20 ng/ml, p = .014) and sICAM-1 (mean ± SD 32.32 ± 6.49 ng/ml versus 24.87 ± 6.79 ng/ml, p < .001) values were found to be significantly higher in pregnant women with PPROM than in healthy pregnant women. A positive and significant correlation was observed between the leukocyte count and the vaginal sVCAM-1 level (r = 0.850; p < .001). CONCLUSION: To the best of our knowledge, this is the first study evaluating the levels of sICAM-1 in maternal serum in pregnant women with PPROM. The maternal serum and vaginal fluid sVCAM-1 and sICAM-1 levels can be used as biochemical markers supporting the PPROM diagnosis because of the increase in both maternal serum and vaginal fluid sVCAM-1 and sICAM-1 levels in pregnant women with PPROM.

Analytical validation of an in-house method for adenosine deaminase determination.

BACKGROUND: The adenosine deaminase (ADA) enzyme is a marker of inflammatory processes whose activity can be measured through a colorimetric method developed as an in-house assay. This validation can reduce costs and expand the alternatives for laboratory diagnosis. METHODS: The ADA analysis was achieved through a modified form of Giusti and Galanti's (1984) method. The following parameters were characterized: calibration curve, linearity, analytical sensitivity, limit of detection, limit of quantification, method working range, precision (within-assay and between-assay), bias, total analytical error, and sample stability. The results were statistically evaluated and compared with quality specifications based on biological variations and the performance of commercial tests. RESULTS: The analytical sensitivity and limit of detection (0.013 and 3.0 U/L, respectively) were lower than those of commercial tests. The method's working range was 3.2-100.0 U/L. According to the quality specification, the method showed optimum performance with a bias <3.5%. However, repeatability (2.2% and 1.7% for normal- and high-activity samples, respectively) and reproducibility achieved worse results when compared to commercial tests. The method demonstrated an inappropriate between-assay precision for low enzymatic activity (10.4%) and the minimum and desirable performance for medium (8.8%) and high (5.0%) activities, respectively. It also presented at least a minimum performance (<25%) for the total analytical error of the three analyzed samples. The pleural fluid samples were found to be stable at -20°C for six days. CONCLUSION: The findings show that the in-house method displays an acceptable performance and is capable of generating results comparable to existing commercial tests.

Two-point measurement of amylase in drainage fluid predicts severe postoperative pancreatic fistula after gastric cancer surgery.

BACKGROUND: Early identification of patients at risk of postoperative pancreatic fistula (POPF) allows appropriate management after gastrectomy. Although some reports have suggested a correlation between POPF and the concentration of amylase in drained abdominal fluid (D-AMY), this has not been proven to impact sufficiently on clinical decision-making. A sustained high level of D-AMY is often assumed to be due to unsatisfactory drainage or excessive pancreatic leakage. We assessed the clinical utility of measuring D-AMY on postoperative day (POD) 1 and POD3 for prediction of POPF. METHODS: Starting in April 2014, 801 patients who underwent radical gastrectomy with prophylactic drain placement were consecutively enrolled. We routinely measured D-AMY on POD1 and POD3, and compared the incidence of problematic POPF and clinical factors including D-AMY. We also attempted to clarify whether such two-point D-AMY measurement was clinically useful for patient management after gastrectomy. RESULTS: Fifty-one of the patients (6.4%) developed Clavien-Dindo grade III or worse POPF. Using D-AMY cutoffs of 2218 IU/L on POD1 and 555 IU/L on POD3, the patients were successfully classified. The highest risk group, in which D-AMY was higher than the cut-off value on both POD1 and POD3, showed a significantly high rate of occurrence (33/105, 31.4%) and high positive likelihood ratio (6.74). Multivariate analysis showed that classification into this highest risk group was an independent risk factor for development of severe POPF (odds ratio 15.2, 95% CI 7.92-29.0). CONCLUSION: Two-point measurement of D-AMY may be an efficient tool for achieving individualized management of POPF following radical gastrectomy.

Development of a selected reaction monitoring mass spectrometry-based assay to detect asparaginyl endopeptidase activity in biological fluids.

Cancer Biomarkers have the capability to improve patient outcomes. They have potential applications in diagnosis, prognosis, monitoring of disease progression and measuring response to treatment. This type of information is particularly useful in the individualisation of treatment regimens. Biomarkers may take many forms but considerable effort has been made to identify and quantify proteins in biological fluids. However, a major challenge in measuring protein in biological fluids, such as plasma, is the sensitivity of the assay and the complex matrix of proteins present. Furthermore, determining the effect of proteases in disease requires measurement of their activity in biological fluids as quantification of the protein itself may not provide sufficient information. To date little progress has been made towards monitoring activity of proteases in plasma. The protease asparaginyl endopeptidase has been implicated in diseases such as breast cancer, leukaemia and dementia. Here we describe a new approach to sensitively and in a targeted fashion quantify asparaginyl endopeptidase activity in plasma using a synthetic substrate peptide protected from nonspecific hydrolysis using D-amino acids within the structure. Our selected reaction monitoring approach enabled asparaginyl endopeptidase activity to be measured in human plasma with both a high dynamic range and sensitivity. This manuscript describes a paradigm for future development of assays to measure protease activities in biological fluids as biomarkers of disease.

Quantification of Human Kallikrein-Related Peptidases in Biological Fluids by Multiplatform Targeted Mass Spectrometry Assays.

Human kallikrein-related peptidases (KLKs) are a group of 15 secreted serine proteases encoded by the largest contiguous cluster of protease genes in the human genome. KLKs are involved in coordination of numerous physiological functions including regulation of blood pressure, neuronal plasticity, skin desquamation, and semen liquefaction, and thus represent promising diagnostic and therapeutic targets. Until now, quantification of KLKs in biological and clinical samples was accomplished by enzyme-linked immunosorbent assays (ELISA). Here, we developed multiplex targeted mass spectrometry assays for the simultaneous quantification of all 15 KLKs. Proteotypic peptides for each KLK were carefully selected based on experimental data and multiplexed in single assays. Performance of assays was evaluated using three different mass spectrometry platforms including triple quadrupole, quadrupole-ion trap, and quadrupole-orbitrap instruments. Heavy isotope-labeled synthetic peptides with a quantifying tag were used for absolute quantification of KLKs in sweat, cervico-vaginal fluid, seminal plasma, and blood serum, with limits of detection ranging from 5 to 500 ng/ml. Analytical performance of assays was evaluated by measuring endogenous KLKs in relevant biological fluids, and results were compared with selected ELISAs. The multiplex targeted proteomic assays were demonstrated to be accurate, reproducible, sensitive, and specific alternatives to antibody-based assays. Finally, KLK4, a highly prostate-specific protein and a speculated biomarker of prostate cancer, was unambiguously detected and quantified by immunoenrichment-SRM assay in seminal plasma and blood serum samples from individuals with confirmed prostate cancer and negative biopsy. Mass spectrometry revealed exclusively the presence of a secreted isoform and thus unequivocally resolved earlier disputes about KLK4 identity in seminal plasma. Measurements of KLK4 in either 41 seminal plasma or 58 blood serum samples revealed no statistically significant differences between patients with confirmed prostate cancer and negative biopsy. The presented multiplex targeted proteomic assays are an alternative analytical tool to study the biological and pathological roles of human KLKs.

Early dual drainage combining transpapillary endotherapy and percutaneous catheter drainage in patients with pancreatic fistula associated with severe acute pancreatitis.

BACKGROUND: The development of pancreatic fistula (PF) associated with pancreatic necrosis is of great concern in the management of severe acute pancreatitis (SAP). We expected that early recognition and intervention of PF combined with percutaneous catheter drainage (PCD) for pancreatic infection may improve SAP outcomes. METHODS: Fifteen consecutive patients with SAP were enrolled. Whenever feasible, fine-needle aspiration for fluid collection was performed to determine infection and amylase concentration. For infection and PF with amylase-rich fluid, PCD and transpapillary endotherapy (preferably naso-pancreatic drainage) were carried out as soon as possible. PCD was intensively managed by irrigating the sized-up and multiple large bore catheters. RESULTS: Infected fluid collection and PF were both detected in 13 (86.7%) patients. Pancreatic duct (PD) disruption (n = 6) and organ failure (n = 5) occurred exclusively in patients with amylase-rich collection ≥10,000 U/L. The median timing of PCD and endotherapy was 15.5 and 16.5 days, respectively. No serious complications or mortality resulted from intervention procedures other than stent occlusion in one (6.7%) patient. Surgical intervention due to uncontrollable infection and visceral organ injury was avoided. Fistula closure was achieved in 12 (92.3%) of 13 PF patients with a median duration of 45 days. Disease-related mortality occurred in one (6.7%) patient. CONCLUSION: Amylase-rich fluid collection ≥10,000 U/L may be an indication for further endoscopic investigation of PD disruption. Early dual drainage combining pancreatic endotherapy and PCD is feasible and safe, and may improve treatment outcome.

High throughput screening of cytokines, chemokines and matrix metalloproteinases in wound fluid induced by mammary surgery.

OBJECTIVE: To clarify the composition of wound fluid (WF) and investigate the impact of WF on breast cancer cell lines. METHODS: The proliferation and migration of WF-treated breast cancer cells MDA-MB-231 and MCF-7 were assessed with colony formation test, MTT cell proliferation test and scratch wound test. The quantitative profiles of WF were analyzed using Bio-Plex Pro kits. RESULTS: The proliferation and migration of WF-treated breast cancer cells were significantly higher than that of untreated cells. Fifteen cytokines, 29 chemokines and 9 matrix metalloproteinases (MMPs) were assessed in WF. The concentrations of these factors were influenced by post-surgery days, neoadjuvant chemotherapy (NAC), TNM stage, pathological type and molecular subtype. The WF harvested from patients underwent NAC showed significant higher profiles of interleukin-1ß (IL-1ß), IL-4, IL-6, IL-17F, IL-21, IL-23, IL-25, IL-31, Interferon γ (IFNγ), CD40 ligand (CD40L), tumor necrosis factor α (TNFα), CXCL1, CXCL2, CXCL5, CCL3, CCL7 and CCL20. CONCLUSIONS: Surgery-induced WF promotes the proliferation and migration of breast cancer cells. The composition of WF is influenced by various clinical features and provides potential therapeutic targets to control local recurrence and tumor progression.

Development of highly sensitive and specific mRNA multiplex system (XCYR1) for forensic human body fluids and tissues identification.

The identification of human body fluids or tissues through mRNA-based profiling is very useful for forensic investigations. Previous studies have shown mRNA biomarkers are effective to identify the origin of biological samples. In this study, we selected 16 tissue specific biomarkers to evaluate their specificities and sensitivities for human body fluids and tissues identification, including porphobilinogen deaminase (PBGD), hemoglobin beta (HBB) and Glycophorin A (GLY) for circulatory blood, protamine 2 (PRM2) and transglutaminase 4 (TGM4) for semen, mucin 4 (MUC4) and human beta defensin 1(HBD1) for vaginal secretion, matrix metalloproteinases 7 and 11 (MMP7 and MMP11) for menstrual blood, keratin 4(KRT4) for oral mucosa, loricrin (LOR) and cystatin 6 (CST6) for skin, histatin 3(HTN3) for saliva, statherin (STATH) for nasal secretion, dermcidin (DCD) for sweat and uromodulin (UMOD) for urine. The above mentioned ten common forensic body fluids or tissues were used in the evaluation. Based on the evaluation, a reverse transcription (RT) PCR multiplex assay, XCYR1, which includes 12 biomarkers (i.e., HBB, GLY, HTN3, PRM2, KRT4, MMP11, MUC4, DCD, UMOD, MMP7, TGM4, and STATH) and 2 housekeeping genes [i.e., glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and 18SrRNA], was developed. This assay was further validated with real casework samples and mock samples (with both single source and mixture) and it was approved that XCYR1 is effective to identify common body fluids or tissues (i.e., circulatory blood, saliva, semen, vaginal secretion, menstrual blood, oral mucosa, nasal secretion, sweat and urine) in forensic casework samples.

Impact of bacterial culture positivity of the drainage fluid during the early postoperative period on the development of intra-abdominal abscesses after gastrectomy.

PURPOSE: The aim of this study was to evaluate the impact of positive bacterial cultures of the drainage fluid (D-cultures) during the early postoperative period on the incidence of intra-abdominal abscess formation following gastrectomy. METHODS: From January 2012 to June 2013, we prospectively performed D-cultures on postoperative day (POD) 1 in consecutive gastric cancer patients who underwent gastrectomy. The univariate and multivariate analyses were performed to identify the risk factors for intra-abdominal abscess formation without anastomotic leakage. RESULTS: The rate of positive D-cultures was 6.4 % on POD 1. According to a univariate analysis, the use of combined organ resection (P = 0.011), the drain amylase level on POD 1 (P = 0.016) and the D-culture status on POD 1 (P = 0.004) were found to be significantly associated with the incidence of intra-abdominal abscesses. A multivariate analysis demonstrated that D-culture positivity on POD 1 was the only independent predictor of intra-abdominal abscess formation (P = 0.011). CONCLUSIONS: The present study demonstrated that bacterial culture positivity of drainage fluid during the early postoperative period has a significant impact on the development of intra-abdominal abscesses after gastrectomy.

Body fluid analysis: clinical utility and applicability of published studies to guide interpretation of today's laboratory testing in serous fluids.

Requests for testing various analytes in serous fluids (e.g., pleural, peritoneal, pericardial effusions) are submitted daily to clinical laboratories. Testing of these fluids deviates from assay manufacturers' specifications, as most laboratory assays are optimized for testing blood or urine specimens. These requests add a burden to clinical laboratories, which need to validate assay performance characteristics in these fluids to exclude matrix interferences (given the different composition of body fluids) while maintaining regulatory compliance. Body fluid testing for a number of analytes has been reported in the literature; however, understanding the clinical utility of these analytes is critical because laboratories must address the analytic and clinical validation requirements, while educating clinicians on proper test utilization. In this article, we review the published data to evaluate the clinical utility of testing for numerous analytes in body fluid specimens. We also highlight the pre-analytic and analytic variables that need to be considered when reviewing published studies in body fluid testing. Finally, we provide guidance on how published studies might (or might not) guide interpretation of test results in today's clinical laboratories.

Chronic venous leg ulcers are associated with high levels of metalloproteinases-9 and neutrophil gelatinase-associated lipocalin.

Venous ulcers are related to dysfunctions in extracellular matrix. Both matrix metalloproteinases (MMP) and neutrophil gelatinase-associated lipocalin (NGAL) could play a role in the healing process in patients with chronic venous ulcers. We evaluated the role of MMP-9 and NGAL in the healing process in venous ulceration. We performed an open-label, parallel groups, single clinical center study. Patients with chronic venous leg ulcers represented the test group (Group I), whereas patients without chronic ulcers represented the control group (Group II). In Group I plasma and wound fluid samples were collected at the time of admission, at the time of the surgery, and at the follow-up, while ulcer tissues were taken at the time of the surgery. In Group II, plasma and wound fluid were collected at admission and at the time of the surgery, whereas skin tissues were collected at the time of the surgery. Enzyme-linked immunosorbent assay test was used to evaluate the levels of MMP-9 and NGAL in plasma and wound fluid, whereas Western blot analysis was performed to estimate the expression of MMP-9 and NGAL in tissues. Enzyme-linked immunosorbent assay tests revealed significantly higher levels of MMP-9 and NGAL in both plasma and wound fluid of patients with ulcers compared to patients without ulcers (p < 0.01). Moreover, Western blot analysis documented an increased expression of MMP-9 and NGAL in biopsy tissue of patients with ulcers compared to patients without ulcers (p < 0.01). In conclusion MMP-9 and NGAL may correlate with the clinical course of venous ulcers.

Release of EPA and DHA from salmon oil - a comparison of in vitro digestion with human and porcine gastrointestinal enzymes.

In the present study, we hypothesised whether in vitro digestion of salmon oil would release different amounts of PUFA depending on the origin of the lipolytic enzymes used. For this purpose, in vitro digestion of salmon oil (SO) was performed using human duodenal juice (HDJ) or a commercial enzyme preparation consisting of porcine pancreatin and bile (PB). The lipolytic effect was determined by measuring the release of fatty acids (FA) using solid-phase extraction and GC-flame ionisation detection, withdrawing samples every 20 min during digestion. The amount of FA released indicated that a plateau was reached after 80 min with approximately similar amounts of FA detected using both HDJ and PB (379 (sd 18) and 352 (sd 23) mg/g SO, respectively). However, the release of 18 : 2, EPA (20 : 5) and DHA (22 : 6) was significantly different during in vitro digestion. At 80 min, HDJ and PB released 43 and 33% of 18 : 2, 14 and 9% of EPA and 11 and 9% of DHA, respectively. Both enzyme preparations released approximately the same amounts of the other FA analysed. The effect of the addition of bile salts (BS) was significantly different in the two enzyme systems, where porcine pancreatin highly responded to the increase in BS concentration, in contrast to HDJ.

Proteolysis of complement factors iC3b and C5 by the serine protease prostate-specific antigen in prostatic fluid and seminal plasma.

Prostate-specific Ag (PSA) is a serine protease that is expressed exclusively by normal and malignant prostate epithelial cells. The continued high-level expression of PSA by the majority of men with both high- and low-grade prostate cancer throughout the course of disease progression, even in the androgen-ablated state, suggests that PSA has a role in the pathogenesis of disease. Current experimental and clinical evidence suggests that chronic inflammation, regardless of the cause, may predispose men to prostate cancer. The responsibility of the immune system in immune surveillance and eventually tumor progression is well appreciated but not completely understood. In this study, we used a mass spectrometry-based evaluation of prostatic fluid obtained from diseased prostates after removal by radical prostatectomy to identify potential immunoregulatory proteins. This analysis revealed the presence of Igs and the complement system proteins C3, factor B, and clusterin. Verification of these findings by Western blot confirmed the high-level expression of C3 in the prostatic fluid and the presence of a previously uncharacterized C-terminal C3 cleavage product. Biochemical analysis of this C3 cleavage fragment revealed a putative PSA cleavage site after tyrosine-1348. Purified PSA was able to cleave iC3b and the related complement protein C5. These results suggest a previously uncharacterized function of PSA as an immunoregulatory protease that could help to create an environment hospitable to malignancy through proteolysis of the complement system.

The effect of smoking on expression of IL-6 and antioxidants in pancreatic fluids and tissues in patients with chronic pancreatitis.

OBJECTIVES: The present study was conducted to ascertain whether cigarette smoke induces oxidative stress in the human pancreas concurrently with inflammation. MATERIALS AND METHODS: The influence of tobacco smoking on the serum level of interleukin-6 (IL-6), on the activities of glutathione peroxidase (GPx) and copper-zinc superoxide dismutase (Cu/Zn SOD) as well as on the metallothionein (MT) level in the pancreatic pseudocyst fluid and its immunohistochemical localization in tissues of non-smoking (n = 9) and smoking (n = 12) patients with diagnosed chronic pancreatitis (CP) was measured. The concentration of interleukin-6 and metallothionein was determined by means of ELISA and the radioisotopic method, respectively. The enzyme activities in the fluid were assayed by the colorimetric method. Samples of tissues of normal pancreas (n = 4) and CP (non-smoking n = 7; smoking n = 12) were verified histopathologically and then IL-6, MT and enzymes were localized by immunohistochemical staining using the monoclonal anti-human antibody. RESULTS: The concentrations of metallothionein and interleukin-6 were significantly higher in smoking patients with CP (as compared with the non-smoking population (p < 0.01; p < 0.001). Interestingly, the ratio of MT/IL-6 in smoking patients with CP was reduced in comparison to non-smoking patients (respectively: 1.1; 5.6). In smoking patients, a significant elevation of the Cu/Zn SOD and GPx activities was revealed as compared with the non-smokers (p < 0.04; p < 0.0007). These studies clearly demonstrate a moderate and strong expression of IL-6 and enzymes in acinar, islet and duct cells of smoking patients. CONCLUSIONS: These observations favor the role of the oxidative stress in the induction of pancreatitis associated with chronic cigarette smoke inhalation.

Insulin complexes with PEGylated basic oligopeptides.

Biodegradable oligolysine and oligoarginine-type homopeptides functionalized with PEG of two different molecular weights interact with insulin, at physiological pH, affording complexes studied by dynamic light scattering, ζ-potential, circular dichroism, FTIR spectroscopy, and isothermal titration calorimetry (ITC). High levels of insulin complexation efficiencies (>99.5%) were determined for all derivatives. FTIR spectra suggest that the positively charged homo-oligopeptide derivatives interact with B chain C-terminus of insulin leading to the formation of nanoparticles than can be traced even at low oligopeptide/insulin molar ratios. The ITC profiles are complex, displaying significant endothermic and exothermic contributions. Oligoarginine-type derivatives exhibit the strongest interactions, while PEGylation of either oligopeptide with the high molecular weight chains significantly affects the ITC profiles and leads to larger enthalpy changes. This may be attributed to PEG-induced aggregation of insulin due to the depletion attraction effect leading to the formation of stable nanocomplexes. Stabilization of complexed insulin against enzymatic degradation by trypsin and α-chymotrypsin is observed especially for the high molecular weight PEGylated arginine-based derivative. Insulin release rates in simulated intestinal fluid are controlled by the length of PEG chains and the presence of arginine end-groups. Released insulin retains its secondary structure as established by circular dichroism spectroscopy.

Novel peptidoglycan-based diagnostic devices for detection of wound infection.

Detection of wound infection is based on evaluation of the well-known signs of inflammation like rubor (redness), calor (heat), tumor (swelling), and dolor (pain) by medical doctors and/or time-consuming procedures requiring special machinery. There is currently no rapid diagnostic device available for the indication of wound infection, which would especially be helpful in home care of chronic ulcer patients. In this study, a new concept for a fast diagnostic tool for wound infection based on lysozyme and elastase triggered release of dye from a peptidoglycan matrix was investigated. The matrix consisted of alginate/agarose and peptidoglycan covalently labeled with Remazol brilliant blue. Lysozyme activity in postoperative wounds and decubitus wound fluids was significantly elevated upon infection (4830 ± 1848 U mL(-1)) compared to noninfected wounds (376 ± 240 U mL(-1)). Consequently, incubation of 8% (w/v) labeled agarose/peptidoglycan blend layers with infected wound fluid samples for 2 h at 37 °C resulted in a 4-fold higher amount of dye released than measured for noninfected wounds. For alginate/peptidoglycan beads, a 7-fold higher amount of dye was released in case of infected wound fluid samples compared to noninfected ones. Apart from lysozyme, proteases [i.e., gelatinase matrix metalloproteinase MMP-2 and MMP-9 and elastase] were detected in wound fluids (e.g., using Western blotting). When dosed in ratios typical for wounds, a slight synergistic effect was measured for peptidoglycan hydrolysis (i.e., dye release) between lysozyme and these proteases. Incubation of a double-layer system consisting of stained and nonstained peptidoglycan with infected wound fluids resulted in a color change from yellow to blue, thus allowing simple visual detection of wound infection.

Exoglycosidase markers of diseases.

Exoglycosidases are hydrolases involved in lysosomal degradation of oligosaccharide chains of glycoconjugates (glycoproteins, glycolipids and proteoglycans). In tissues and body fluids, a higher exoglycosidase specific activity is found in N-acetyl-ß-hexosaminidase, than ß-glucuronidase, α-L-fucosidase, ß-galactosidase, α-mannosidase and α-glucosidase. Determination of exoglycosidases (especially N-acetyl-ß-hexosaminidase and ß-glucuronidase) in body fluids could be an inexpensive, easy to perform and sensitive test for pathological evaluation, as well as in screening and monitoring many diseases, including alcohol abuse, risk of arteriosclerosis, bacterial infections (e.g. Lyme borreliosis), chronic inflammatory processes, such as rheumatoid arthritis and juvenile idiopathic arthritis, asthma, autoimmune hepatitis and primary biliary cirrhosis, as well as cancers.

Cystatin A protease inhibitor and cysteine proteases in human cervicovaginal fluid in term pregnancy and labor.

OBJECTIVE: The purpose of this study was to investigate the temporal changes in immunoreactive cystatin A and the enzymatic activity of cathepsins B, H, L, and S in human cervicovaginal fluid (CVF) in late pregnancy and spontaneous labor. STUDY DESIGN: CVF was collected weekly (n = 95 women) from 36 weeks gestation until spontaneous term labor. Cystatin A was quantified using enzyme-linked immunosorbent assay. The enzyme activity of cathepsins B, H, L, and S was measured with fluorometric enzyme assay kits. RESULTS: Cystatin A significantly decreased towards (P = .016, 2-way analysis of variance) and during labor (P < .001, 2-way analysis of variance). Enzymatic activity of cathepsins B, H, and S did not change with labor onset (P = .452, P = .703, P = .411, respectively, 2-way analysis of variance). CONCLUSION: In late gestation, CVF-decreased expression of the cysteine protease inhibitor, cystatin A, is associated with labor. Although the role and contribution of cystatin A to increased extracellular matrix remodeling has yet to be elucidated, the data that were obtained are consistent with this hypothesis.

Analytical performances of the Diazyme ADA assay on the Cobas® 6000 system.

OBJECTIVE: To evaluate the analytical performance of the Diazyme ADA assay on the Cobas® 6000 system for pleural fluid samples analysis. DESIGN AND METHODS: Imprecision, linearity, calibration curve stability, interference, and correlation studies were completed. RESULTS: The Diazyme ADA assay demonstrated excellent precision (CV<4%) over the analytical measurement range (0.5-117 U/L). Bilirubin above 50 µmol/L and haemoglobin above 177 µmol/L interfered with the test, inducing a negative and a positive interference respectively. The Diazyme ADA assay correlated well with the Giusti method (r(2)=0.93) but exhibited a negative bias (~ -30%). CONCLUSIONS: The Diazyme ADA assay on the Cobas® 6000 system represents a rapid, accurate, precise and reliable method for determination of ADA activity in pleural fluid samples.

Insulin regulates alveolar epithelial function by inducing Na+/K+-ATPase translocation to the plasma membrane in a process mediated by the action of Akt.

Stimulation of Na(+)/K(+)-ATPase translocation to the cell surface increases active Na(+) transport, which is the driving force of alveolar fluid reabsorption, a process necessary to keep the lungs free of edema and to allow normal gas exchange. Here, we provide evidence that insulin increases alveolar fluid reabsorption and Na(+)/K(+)-ATPase activity by increasing its translocation to the plasma membrane in alveolar epithelial cells. Insulin-induced Akt activation is necessary and sufficient to promote Na(+)/K(+)-ATPase translocation to the plasma membrane. Phosphorylation of AS160 by Akt is also required in this process, whereas inactivation of the Rab GTPase-activating protein domain of AS160 promotes partial Na(+)/K(+)-ATPase translocation in the absence of insulin. We found that Rab10 functions as a downstream target of AS160 in insulin-induced Na(+)/K(+)-ATPase translocation. Collectively, these results suggest that Akt plays a major role in Na(+)/K(+)-ATPase intracellular translocation and thus in alveolar fluid reabsorption.