Search

Tuberculosis (TB) is the leading infectious cause of death worldwide, and the commonest cause of death in people living with HIV. Globally, pleural TB remains one of the most frequent causes of pleural exudates, particularly in TB-endemic areas and in the HIV positive population. Most TB pleural effusions are exudates with high adenosine deaminase (ADA), lymphocyte-rich, straw-coloured and free flowing, with a low yield on mycobacterial culture. TB pleurisy can also present as loculated neutrophil-predominant effusions which mimic parapneumonic effusions. Rarely, they can present as frank TB empyema, containing an abundance of mycobacteria. Up to 80% of patients have parenchymal involvement on chest imaging. The diagnosis is simple if M. tuberculosis is detected in sputum, pleural fluid or biopsy specimens, and the recent advent of liquid medium culture techniques has increased the microbiological yield dramatically. Where the prevalence of TB is high the presence of a lymphocyte-predominant exudate with a high ADA has a positive predictive value of 98%. In low prevalence areas, the absence of an elevated ADA and lymphocyte predominance makes TB very unlikely, and pleural biopsy should be performed to confirm the diagnosis. Pleural biopsy for liquid culture and susceptibility testing must also be considered where the prevalence of drug resistant TB is high. Treatment regimens are identical to those administered for pulmonary TB. Initial pleural drainage may have a role in symptom relief and in hastening the resolution of the effusion. Surgical intervention may be required in loculated effusions and empyemas., (© 2018 John Wiley & Sons Ltd.)

Introduction: Pleural fluid adenosine deaminase level can be applied to rapidly detect tuberculous pleural effusion. We aimed to establish a local diagnostic cut-off value for pleural fluid adenosine deaminase to identify patients with tuberculous pleural effusion, and optimise its utility., Methods: We retrospectively reviewed the medical records of consecutive adults with pleural fluid adenosine deaminase level measured by the Diazyme commercial kit (Diazyme Laboratories, San Diego [CA], United States) during 1 January to 31 December 2011 in a cluster of public hospitals in Hong Kong. We considered its level alongside early (within 2 weeks) findings in pleural fluid and pleural biopsy, with and without applying Light's criteria in multiple scenarios. For each scenario, we used the receiver operating characteristic curve to identify a diagnostic cut-off value for pleural fluid adenosine deaminase, and estimated its positive and negative predictive values., Results: A total of 860 medical records were reviewed. Pleural effusion was caused by congestive heart failure, chronic renal failure, or hypoalbuminaemia caused by liver or kidney diseases in 246 (28.6%) patients, malignancy in 198 (23.0%), non-tuberculous infection in 168 (19.5%), tuberculous pleural effusion in 157 (18.3%), and miscellaneous causes in 91 (10.6%). All those with tuberculous pleural effusion had a pleural fluid adenosine deaminase level of ≤100 U/L. When analysis was restricted to 689 patients with pleural fluid adenosine deaminase level of ≤100 U/L and early negative findings for malignancy and non-tuberculous infection in pleural fluid, the positive predictive value was significantly increased and the negative predictive value non-significantly reduced. Using this approach, neither additionally restricting analysis to exudates by Light's criteria nor adding closed pleural biopsy would further enhance predictive values. As such, the diagnostic cut-off value for pleural fluid adenosine deaminase is 26.5 U/L, with a sensitivity of 87.3%, specificity of 93.2%, positive predictive value of 79.2%, negative predictive value of 96.1%, and accuracy of 91.9%. Sex, age, and co-morbidity did not significantly affect prediction of tuberculous pleural effusion using the cut-off value., Conclusion: We have established a diagnostic cut-off level for pleural fluid adenosine deaminase in the diagnosis of tuberculous pleural effusion by restricting analysis to a level of ≤100 U/L, and considering early pleural fluid findings for malignancy and non-tuberculous infection, but not Light's criteria.

Disease processes may impair the production and reabsorption of fluid from in the body cavities, which results in its excessive accumulation., Aim: The aim of the study was the evaluation of difficulties regarding the classification of fluids from the body cavities into transudate/exudate observing the following: Light's criteria, total fluid protein concentration, and total protein ratio (TP ratio) and lactate dehydrogenase ratio (LDH ratio)., Materials and Methods: Retrospective analysis was conducted on pleural (N=314), peritoneal (N=114) and pericardial (N=10) fluids, which were tested for the total protein concentration and LDH activity both in fluid and serum and calculated on TP ratio and LDH ratio., Results: Based on the total protein concentration, 278 fluids from pleural cavity were classified as an exudate; 36 as a transudate. Applying the Light's criteria 240 fluids were classified as an exudate; the remaining 74 fluids were classified as a transudate. Based on TP and LDH ratios, 229 fluids from pleural cavity were classified as an exudate; 85 as a transudate. Depending on the total protein concentration, 35 fluids from the peritoneal cavity were classified as an exudate; 79 as a transudate. Applying the Light's criteria 54 fluids were classified as an exudate; the remaining 60 fluids were classified as a transudate. Based on TP and LDH ratios, 22 fluids from peritoneal cavity were classified as an exudate; 92 as a transudate. Analysis of pericardial fluids, depending on the total protein concentration classified 9 of them as an exudate and 1 as a transudate. The same results were obtained by applying Light's criteria. Based on TP and LDH ratios, 7 fluids from pericardial cavity were classified as an exudate; 3 - as a transudate., Conclusions: Applying the Light's criteria or the total protein concentration in differential diagnostics of fluids from the body cavities resulted in qualification more of them as an exudates as compared to the analysis of the same fluids depending on the TP and LDH ratios. It can be assumed that some of the transudative/exudative fluids were incorrectly classified. Performed analysis suggest that more adequate criteria of the classification of fluids from the body cavities into transudate/exudate are of great importance.

Objective: To determine the matrix metalloproteinase-2 (MMP-2) levels in root-canal exudates from teeth undergoing root-canal treatment., Material and Methods: The root-canal exudates from six teeth with normal pulp and periradicular tissues that required intentional root canal treatment for prosthodontic reasons and from twelve teeth with pulp necrosis and asymptomatic apical periodontitis (AAP) were sampled with paper points for bacterial culture and aspirated for the detection of proMMP-2 and active MMP-2 by gelatin zymography and the quantification of MMP-2 levels by ELISA., Results: By gelatin zymography, both proMMP-2 and active MMP-2 were detected in the first collection of root-canal exudates from teeth with pulp necrosis and AAP, but not from teeth with normal pulp, and their levels gradually decreased and disappeared at the last collection. Consistently, ELISA demonstrated a significant decrease in MMP-2 levels in the root-canal exudates of teeth with pulp necrosis and AAP following root canal procedures (p<0.05). Furthermore, the MMP-2 levels were significantly lower in the negative bacterial culture than those in the positive bacterial culture (p<0.001)., Conclusions: The levels of MMP-2 in root-canal exudates from teeth with pulp necrosis and AAP were gradually reduced during root canal procedures. Future studies are required to determine if MMP-2 levels may be used as a biomolecule for the healing of apical lesions, similar to the clinical application of MMP-8 as a biomarker., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Introduction: Although pleural effusion (PE) can be caused by several pathologies like congestive heart failure, infections, malignancies, and pulmonary embolism, it is also a common finding in chronic kidney disease (CKD). Diagnostic thoracentesis is of limited value in the differential diagnosis, and the role of more invasive investigations like medical thoracoscopy (MT) is still unclear., Aim: To evaluate the usefulness of MT in unexplained PE in CKD., Materials and Methods: In the electronic database of our Institution, we retrospectively searched for patients with CKD who underwent MT for unexplained PE between January 2008 and August 2016., Results: Ten patients were included in the present study. The average age was 72.4 years, the male:female ratio 9:1 and the average blood creatinine value 5.96 mg/dL. The average follow-up was 18 months.A thoracentesis showed an exudate was found in 9 patients and in 1 case pleural fluid characteristics were not recorded for technical reasons; in none of them the cytologic or microbiological analyses were considered diagnostic.The clinical suspicion was a neoplastic (5) or an infectious disease (5). In 4 patients with recurrent PE, MT was performed to obtain talc pleurodesis.No immediate procedure-related complications were recorded; 1 patient developed empyema after 2 months. In 6 cases final diagnosis was chronic uremic pleuritis, hydrothorax in 2, and chronic lymphocytic pleurisy in 2., Conclusions: MT represents a safe and effective diagnostic and therapeutic procedure in patients with CKD, that itself is a common cause of exudative effusion, and those patients may not require MT.

Post-operative pancreatic fistula (POPF) is a potentially severe complication following distal pancreatectomy. The aim of this study was to assess the predictive value of intra-operative amylase concentration (IOAC) in peri-pancreatic fluid after distal pancreatectomy for the diagnosis of POPF. Consecutive patients who underwent a distal pancreatectomy between November 2014 and September 2016 were included in the analysis. IOAC was measured, followed by drain fluid analysis for amylase on post-operative days (PODs) 1, 3, and 5. Receiver operator characteristic (ROC) analysis was performed to evaluate the discriminative capacity of IOAC as a predictor of POPF. IOAC was measured after distal pancreatectomy in 26 patients. The IOAC correlated significantly with (i) PODs 1, 3, and 5 drain amylase (p < 0.01); (ii) the development of POPF (p < 0.01); and (iii) the Clavien-Dindo grade of surgical complications (p = 0.02). Eighty-three percent of patients with an IOAC > 1000 experienced a post-operative complication (OR 18.3, 95% CI 2.51-103, p < 0.01). ROC curve analysis confirmed the predictive relationship of IOAC and POPF as an excellent test with an area under the curve of 0.92 (95% CI 0.81-0.99, p < 0.01). Measurement of IOAC allows early and accurate categorization of patients at risk for POPF in distal pancreatectomy.

Proteases are important for wound healing, but in excessive amounts or left uncontrolled, they may cause healing impairment or other severe wound complications. Point-of-care testing for protease activities in wounds may be useful for monitoring the effectiveness of treatment, and for early identification of wounds that potentially fail to heal. Here we describe an easy, noninvasive method to collect wound fluid for evaluating the protease milieu of wounds. Wound fluids were collected using sterile sponges applied between wound surface and normal wound dressing. Wound fluid could be easily squeezed or centrifuged out of the sponges and was tested for gelatinase (MMP-2 and MMP-9) activities by gel zymography. In addition, we measured polymorphonuclear granulocyte elastase levels by ELISA. Both gelatinases were remarkably stable in sponge derived fluids, as no significant loss was observed even when samples were stored for 3 days at room temperature. Protease levels were highly diverse amongst patients and, in some cases, showed substantial variations in the course of the treatment. The here described wound sponge approach represents a patient-friendly and reliable method to collect wound fluid for evaluating wound healing relevant biomarkers, such as matrix metalloproteinases., (© 2017 by the Wound Healing Society.)

Background: Matrix metalloproteinases (MMPs) are responsible for the breakdown of the extracellular matrix and play an important role in the inflammatory processes of pleural exudates. The imbalance between MMPs and their inhibitors (TIMPs) is present in various pathological processes., Objective: To evaluate the profile of MMPs and TIMPs in pleural effusions of different etiologies correlated with inflammatory markers., Methods: The patients with pleural effusion due to tuberculosis (TB), cancer (CA) or transudate were prospectively evaluated. Pleural fluid was submitted to cytological, biochemical, cytokines, MMP, and TIMP analysis. Statistical analysis was performed using ANOVA and Spearman's correlation, and p < 0.05 was considered significant., Results: One hundred and fourteen patients were enrolled, 80 exudates (41 TB and 39 CA) and 34 transudates. The levels of MMP-8 and MMP-9 were higher in exudates compared to transudates. The level of MMP-8 was significantly higher in TB than in CA. TIMP-1 levels were higher in exudates. IL-6, VEGF, and TGF-β 1 showed differences between exudates and transudates. However, IL-6 level was higher in TB than in CA. We found a significant correlation between MMPs and TIMPs with inflammation markers. MMP-1 was correlated with LDH levels. MMP-8 was correlated with LDH, total cell count, neutrophils, and ADA as well as MMP-1 levels. MMP-9 was correlated with IL-6, TGF-β 1 , and VEGF. TIMP-1 was correlated with MMP-9 and IL-6., Conclusions: MMPs and TIMPs are expressed in pleural fluid of different etiologies and correlate with inflammatory mediators. MMPs may be useful in determining the cause of fluid, but more studies are needed to determine the spectrum of diseases associated with the various isoforms of MMPS and TIMPs.

Background: Lactate dehydrogenase (LDH) activity is often measured in human effusions to help in differentiating between transudates and exudates. Few studies have been performed using effusion samples from animals., Objectives: The purpose of the study was to determine whether LDH can be used to differentiate between transudative and exudative effusions in dogs and cats (including postmortem samples), and whether there is a difference between different laboratory methods of LDH measurement., Methods: Lactate dehydrogenase activity was measured in canine and feline effusions that were submitted to the Murdoch University Veterinary Hospital Clinical Pathology Laboratory over approximately 12 months using 2 wet and one dry chemistry methods, including 10 effusions collected postmortem. Results were compared to classification using traditional methods for effusion types., Results: Lactate dehydrogenase activity was significantly higher in exudates than in transudates, significantly different depending on the method of measurement, and significantly higher in all effusions collected postmortem. An LDH effusion:serum ratio of < 0.5 was associated with transudates. There was no significant difference between samples collected into EDTA or plain serum tubes, in frozen and thawed samples, or after storage at 4°C for 3-7 days., Conclusions: Measurement of LDH activity may be useful in helping to differentiate between transudates and exudates in cats and dogs. The method of measurement must be known and kept consistent if cutoff values are to be used. The LDH activity was increased in all effusions collected from animals after death, potentially invalidating its use postmortem., (© 2016 American Society for Veterinary Clinical Pathology.)

Purpose of Review: This article summarizes current data regarding the accuracy of pleural fluid tests assisting the diagnosis of tuberculous pleuritis (TBP)., Recent Findings: No pleural fluid test reliably rules-in TBP in settings with low TBP prevalence. Interferon-γ) alone or in combination with adenosine deaminase (ADA) is more reliable than ADA for this purpose in nonlow prevalences. ADA can reliably rule-out TBP in prevalences of less than 40% although in higher prevalences the product of interleukin-27 and ADA is the most accurate rule-out test., Summary: The definite diagnosis of TBP requires the isolation of Mycobacterium tuberculosis from pleural fluid or biopsies. Because of the low sensitivity of pleural fluid cultures and the invasiveness of pleural biopsy techniques, the concept of a pleural fluid test that accurately establishes or excludes TBP diagnosis has been proposed. Numerous pleural fluid tests have been evaluated for this purpose with ADA being the most widely accepted one. During the last years, it has been demonstrated that the ability of ADA to rule-in or rule-out TBP is affected by the prevalence of TBP in the setting where the test is used. The complementary use of interferon-γ or interleukin-27 increases the ability of ADA to rule-in or rule-out the disease, respectively.

Chronic venous ulceration (CVU) of the lower limbs is a common condition affecting 1% of the adult population in Western countries, which is burdened with a high complication rate and a marked reduction in the quality of life often due to prolonged healing time. Several metalloproteinases (MMPs) such as MMP-9 together with neutrophil gelatinase-associated lipocalin (NGAL) appear to be involved in the onset and healing phases of venous ulcer, but it is still unclear how many biochemical components are responsible for prolonged healing time in those ulcers. In this study, we evaluate the role of MMP-1 and MMP-8 in long lasting and refractory venous ulcers. In a 2-year period we enroled 45 patients (28 female and 17 male, median age 65) with CVU. The enroled population was divided into two groups: group I were patients with non-healing ulcers (ulcers that had failed to heal for more than 2 months despite appropriate treatments) and group II were patients with healing ulcers (ulcers in healing phases). MMP-1 and MMP-8 were measured in fluids and tissues of healing and non-healing ulcers by means of enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, respectively. In particular the patterns of the collagenases MMP-1 and MMP-8 in healing wounds were distinct, with MMP-8 appearing in significantly greater amounts especially in the non-healing group. Our findings suggest that MMP-1, and MMP-8 are overexpressed in long lasting CVU. Therefore, this dysregulation may represent the main cause of the pathogenesis of non-healing CVU., (© 2013 The Authors. International Wound Journal © 2013 Medicalhelplines.com Inc and John Wiley & Sons Ltd.)

The aims of this study were to investigate the usefulness of LDH measurement in effusions in dogs to classify the fluid as exudate or transudate and to classify the fluid based on the pathophysiological mechanism. In thoracic (n = 107) and abdominal (n = 199) fluid of dogs cell count, protein and LDH concentrations were measured. The fluid was retrospectively categorized into exudate (group A), protein-poor (B) or protein-rich transudate (C) as well as based on pathophysiology into the following five groups (group 1 - 5): hemorrhagic, chylous, inflammatory, oncotic and congestive. In thoracic and abdominal fluid LDH concentrations were significantly higher in group A compared to group B and C. There was a significant difference of LDH concentration between the groups 1 to 5 in both thoracic and abdominal fluid, however there was a large overlap between the five groups. While fluid LDH measurement in dogs is helpful to distinguish exudate from transudate it is only of little help to elucidate the pathophysiological cause.

Background and Aim: The purpose of this study was to assess the relationship of pleural adenosine deaminase (P-ADA) and non-Hodgkin's lymphoma (NHL)., Design and Methods: We retrospectively analysed 63 NHL patients with pleural effusions who accepted a diagnostic thoracentesis and who had P-ADA available at the China Medical University Hospital (Taichung, Taiwan) between January 2003 and April 2012., Results: There were 46 exudates [40 malignant pleural effusions (MPE), 5 complicated para-pneumonic effusions and 1 undiagnosed effusion] and 17 transudates. The P-ADA activity was significantly different between the two groups (P < 0.005). Among 40 MPE cases, 29 were due to B-cell and 11 due to T-cell NHL. There was no pleural transudative effusion with P-ADA value higher than 26 U/l in our study, but simultaneously 48% (22/46) of exudative pleural effusions showed a P-ADA value under that cut-off point. The P-ADA level reached the diagnostic cut-off for tuberculosis (40 IU/l) in 11 cases of MPE (11/40 = 27.5%): 9 B-cell NHL (9/29 = 31%) and 2 T-cell NHL (2/11 = 18%). The median levels (25th, 75th percentiles) of P-ADA were 28 IU/l (14-50) in the MPE of B-cell NHL and 26 IU/l (14-28) in the T-cell NHL (P = 0.693)., Conclusions: The use of P-ADA in NHL effusion could aid the separation of transudates from exudates. Around one-quarter MPE of NHL had abnormal P-ADA ( > 40 IU/l). There was no difference in the P-ADA activity in T-cell and B-cell NHL., (© Crown copyright 2014.)

Flaminal Forte is an enzyme alginogel,whose activity depends on the absorption and binding of matrix metalloproteinases (MMPs), which are known to play a crucial role in delayed wound healing. The aim of the study was to evaluate the influence of Flaminal on MMP-2/-9 activity in ulcer exudate, ex vivo. Eight patients with bilateral venous leg ulcers were treated for 4 weeks with Flaminal Forte covered by hydrocolloid ('F' wounds), or with hydrocolloid alone ('H' wounds) as a reference control. Clinical assessment did not reveal any differences between F and H wounds regarding surface reduction and general wound condition. Nevertheless, although non-significant, there was a visible difference in peri-wound skin appearance in F wounds, as compared to H wounds. The wound exudate contained high MMP-2/-9 levels, which gradually decreased as wounds healed. The attenuation of MMPs was stronger in F than in H exudate, however, in standard zymography this difference appeared non-significant. Real-time zymography revealed that Flaminal mediated a powerful direct inhibition of gelatinolytic activity of wound exudate and of recombinant MMP-2/-9 in vitro.

Background: Adenosine deaminase (ADA) is useful in the diagnosis of tuberculous pleural effusion (TPE). This study aims to determine the factors affecting pleural fluid ADA levels and to establish the optimal ADA levels for diagnosis of TPE for different age groups., Methods: This was a retrospective study from January 2007 to October 2011. One hundred and sixty patients who had pleural fluid ADA performed for investigation of pleural effusion were analyzed. Variables examined included demographics, pleural fluid characteristics and peripheral blood counts. The ADA cut-offs according to age were selected using the receiver operating characteristic (ROC) curve., Results: The mean pleural fluid ADA was significantly higher in the TPE group (100 ± 35 IU/L) compared to non TPE patients (30 ± 37 IU/L). There was significant correlation between pleural fluid ADA and age, pleural fluid protein, LDH, and fluid absolute lymphocyte count. The strongest correlation was seen with age (r = -0.621). For patients ≤ 55 years old the ROC for ADA had area under curve (AUC) of 0.887. A pleural fluid ADA of 72 IU/L had sensitivity of 95.1%, specificity of 87.5%, positive predictive value (PPV) of 95.1% and negative predictive value (NPV) of 87.5% for the diagnosis of TPE. For patients > 55 years old the AUC is 0.959. ADA of 26 IU/L had a sensitivity of 94.7%, specificity of 80.4%, PPV of 62% and NPV of 97.8%., Conclusions: There is a significant negative correlation between pleural fluid ADA and age. For older patients, a lower ADA cut-off should be used to exclude TPE.

Objective: To test the binding capacity of a hydrokinetic fiber dressing for PMN elastase, MMP-2 and MMP-9 in vitro, and to determine whether testing of dressing material samples in vitro is sufficient to predict the performance of the whole dressing., Method: In vitro protease binding assays for PMN elastase, MMP-2 and MMP-9 were used to evaluate the protease modulating capacity of sorbion sachet EXTRA, a superabsorbant dressing that consists of special hydrokinetic fibers, which are formed from cellulose and sodium polyacrylate in a mechanical process without any bonding agents or adhesives. The ability of the hydrokinetic fiber dressing to inhibit elastase and collagenase activity was also tested at 0%, 50% and 100% saturation volume., Results: The hydrokinetic fiber wound dressing was able to bind considerable amounts of elastase, reducing elastase activity by approximately 84%. Moreover, it significantly decreased MMP-2 and MMP-9 concentrations in vitro and was able to completely inhibit collagenase activity., Conclusion: In summary, the hydrokinetic fiber dressing sorbion sachet EXTRA was able to significantly reduce the concentration and activity of proteolytic enzymes in vitro. These results suggest that sorbion sachet EXTRA should have a beneficial action by reducing the detrimental effects of proteolytic enzymes in vivo., Declaration of Interest: This work was supported by Sorbion GmbH & Co. KG, Senden, Germany.

Objective: To demonstrate the binding and inactivation action of a superabsorbent dressing on proteolytic enzymes MMP-2, MMP-9 and collagenase using an established methodology., Method: An in vitro assay of MMP binding and collagenase inactivation has been conducted using the superabsorbent wound dressing (Eclypse; Advancis Medical UK). Dressings in this category, and other absorbents, have been claimed to possess MMP-binding characteristics; however, for most there is no published evidence as yet. In this series of experiments, we have used validated experimental techniques to evaluate such activity., Results: Results show that the superabsorbent dressing does have a statistically-significant effect in binding two of the most important MMPs, MMP-2 and MMP-9, as well as inhibiting collagenase., Conclusion: These results support this activity for the superabsorbent dressing and indicate a probable beneficial clinical action in reducing the influence of these enzymes in delayed wound healing.

Collagen/oxidized regenerated cellulose (ORC)/silver therapy has been designed to facilitate wound healing by normalizing the microenvironment and correcting biochemical imbalances in chronic wounds. The aim of this study was to compare collagen/ORC/silver therapy to control (standard treatment). Patients with diabetic foot ulcers were randomized to either collagen/ORC/silver (24) or control treatment (15). Wound area measurements and wound fluid samples were taken weekly. Protease levels were measured in wound fluid samples to investigate differences between responders (≥50% reduction in wound area by week 4) and nonresponders (<50% reduction in wound area by week 4). There were significantly more responders in the collagen/ORC/silver group compared with the control group (79% vs. 43%, p = 0.035). There were significantly fewer withdrawals from the study because of infection in the collagen/ORC/silver group compared with the control group (0% vs. 31%, p = 0.012). The sum of matrix metalloproteinase-9 and elastase concentration was higher in nonresponders compared with responders at baseline (p = 0.0705) and week 4 (p = 0.012). The results suggest that collagen/ORC/silver normalizes the wound microenvironment and protects against infection, resulting in improved wound healing. It was also demonstrated that measuring a combination of proteases may be a more relevant prognostic healing marker than any individual protease alone., (© 2013 by the Wound Healing Society.)

Pleural adenosine deaminase activity (ADA) is a sensitive and specific test for tuberculous pleurisy. Here, we report a case of undetectable ADA in the pleural fluid of a man presenting with chronic cough, fever and night sweats. Subsequent laboratory investigations and review revealed that the presence of high concentrations of ammonia in pleural fluid, commonly seen in empyema, negatively interferes with ADA results when measured by the Guisti and Galanti method. The source of ammonia may come from deamination of amino acids, ammonia-producing microbes and/or leucocytes. This interference invalidates ADA results and is present in ∼2% of our laboratory requests. It is important to keep this interference in mind when tuberculosis/ammonia-producing bacteria coinfections are suspected and during early phases of tuberculous pleurisy, when neutrophils predominate in the pleural fluids.

Purpose of the Study: Leukocyte esterase is an enzyme in neutrophils from which it is released into exudate; its detection by colorimetric test strips indicates the presence of neutrophils. This is a rapid method to find whether exudate is of infectious or non-infectious aetiology. The aim of the study was to determine the sensitivity and specificity of leukocyte esterase testing with use of AUTION Sticks (Arkray) for examination of exudates obtained in inflammatory diseases of the skeletal system., Material and Methods: Exudates associated with skeletal system diseases were collected from 45 patients in the period from July 1st to December 31 st , 2012. Aspirates obtained under sterile conditions were examined for leukocyte esterase; cytological and microbiological examinations were also carried out. For the detection of leukocyte esterase, a drop of aspirate was placed on the reagent zone of a test strip and the resulting colour reaction was read after 90 minutes. Changes in colour were compared with a reference strip provided by the manufacturer. The results were assessed on a five-shade scale as follows: 0, no colour change; 1 to 4, gradual change from light pink to deep purple. The results were compared with those of cytological and microbiological examinations. Shade 4 on the strip corresponded to a positive cytological finding of bacterial infection, and shades 3 and 4 correlated with a positive microbial finding. The sensitivity and specificity of leukocyte esterase testing were statistically evaluated for both comparisons., Results: Based on the results of cytological and microbiological examinations, an infectious aetiology of exudate was diagnosed in 21 (44.4%) and non-infectious aetiology in 24 (63.6%) patients. With leukocyte esterase reagent strips when shade 4 was taken as a positive result, the sensitivity and specificity of examination was assessed as 0.6190 and 0.9583, respectively. When taking both shade 3 and shade 4 for a positive result, sensitivity and specificity were 0.8571 and 0.8750, respectively. Shades 0 and 1 corresponded to the number of leukocytes in exudate that was no higher than 2 x 10⁹/ml., Discussion: The detection of leukocyte esterase is a quick and easy examination. It is useful for readily excluding or confirming an infectious aetiology of exudate and can, to some extent, substitute a cytological examination. It can also help to make a quick decision whether one- or two-stage joint reimplantation should be performed and thus eliminate the need of intra-operative histological examination of frozen tissue samples. A drawback of the method was that exudate samples contaminated with blood interfered with an assessment of colour shades. However, this can be avoided by centrifugation of the sample and use of a supernatant free from erythrocytes., Conclusions: Diagnosing infectious aetiology of joint exudate or exudate from an abscess using leukocyte esterase reagent strips appears, according to our results, to be a promising, semi-quantitative method with high specificity and sensitivity which is rapid, simple and affordable. It can be useful particularly in out-patient institutions for a quick diagnosis of arthritis; intraoperatively, it can serve as an additional method to other exudate examinations.

Tuberculosis (TB) is a major public health problem in developing countries including Nepal. One of the common presentations of TB is pleural effusion. The diagnosis of tubercular pleural effusion can be difficult because of the low rate of detecting tubercule bacilli by direct stain and culture of pleural fluid for acid-fast bacilli (AFB). Pleural biopsy can be useful but is invasive and requires experts. In this context, pleural fluid Adenosine Deaminase (ADA) level has been proposed as easy, cheap and highly sensitive test for diagnosis of TB pleural effusion. The present study was undertaken to define the role ofpleural fluid ADA value in accurate diagnosis of TB pleural effusion. A Prospective analysis of 100 patients admitted in Nepal Medical College and teaching Hospital with pleural effusion was done. Pleural fluid ADA level was evaluated in all patients, and significance of pleural fluid ADA level in TB pleural effusion was studied. It was found that mean ADA level in pleural fluid was 105.8 +/- 67.23 U/L in cases of TB, as compared to 16.83 +/- 8.91 U/L in malignancy, 44.53 +/- 32.84 U/L in parapneumonic effusion and 15.94 +/- 4.88 U/L in patients with miscellaneous diagnosis. For a cut-off value of 42.19 U/L for diagnosis of TB pleural effusion, sensitivity was found to be 90.8% and specificity 82.8%. Almost all patients diagnosed to have TB pleural effusion responded completely to anti-tubercular treatment. So, we concluded that pleural fluid ADA analysis could be easy, cheap and highly sensitive and specific test for diagnosis of TB pleural effusion.

Cutaneous wound healing is orchestrated by a number of physiological pathways that ultimately lead to reformation of skin integrity and the production of functional scar tissue. The remodeling of a wound is significantly affected by matrix metalloproteinases (MMPs), which act to control the degradation of the extracellular matrix (ECM). Regulation of MMPs is imperative for wound healing as excessive levels of MMPs can lead to disproportionate destruction of the wound ECM compared to ECM deposition. In addition to human MMPs, bacterial proteases have been found to be influential in tissue breakdown and, as such, have a role to play in the healing of infected wounds. For example, the zinc-metalloproteinase, elastase, produced by Pseudomonas aeruginosa, induces degradation of fibroblast proteins and proteoglycans in chronic wounds and has also been shown to degrade host immune cell mediators. Microbial extracellular enzymes have also been shown to degrade human wound fluid and inhibit fibroblast cell growth. It is now being acknowledged that host and bacterial MMPs may act synergistically to cause tissue breakdown within the wound bed. Several studies have suggested that bacterial-derived secreted proteases may act to up-regulate the levels of MMPs produced by the host cells. Together, these findings indicate that bacterial phenotype in terms of protease producing potential of bacteria should be taken into consideration during diagnostic and clinical intervention of infected wound management. Furthermore, both host MMPs and those derived from infecting bacteria need to be targeted in order to increase the healing capacity of the injured tissue. The aim of this review is to investigate the evidence suggestive of a relationship between unregulated levels of both host and bacterial proteases and delayed wound healing., (© 2012 by the Wound Healing Society.)

Background and Objective: The aim of this study was to determine whether high levels of pleural adenosine deaminase (pADA) are predictive for tuberculosis when pleural effusions do not satisfy the criteria for lymphocytic effusions or show neutrophil predominance., Methods: This was a retrospective observational study of 147 consecutive patients with exudative pleural effusions that were diagnosed by analysis of fluid samples during a 3-year period from 1 April 2007 to 31 March 2010. Multiple linear correlation tests were used to assess clinical variables as possible predictors of high pADA levels., Results: High pleural LDH (pLDH) and pleural potassium (pK) levels were associated with high pADA levels (P < 0.0001). Although there was a linear correlation between pLDH and pADA levels in patients with parapneumonic effusions (PPE) (n = 75), tubercular effusions (n = 21), malignant effusions (n = 41) and miscellaneous effusions (n = 10), a significant linear correlation between pK and pADA levels was observed only in patients with PPE (ρ = 0.525, P < 0.0001). When the cut-off value for pK was set at 5.0 mEq/L, pADA levels were >50 IU/L and pK levels were >5.0 mEq/L in only one patient (5%) in the tuberculosis group (n = 21) and 15 patients (12%, all with PPE) in the non-tuberculosis group (n = 126)., Conclusions: When pK levels exceed 5.0 mEq/L, high pADA levels do not necessarily indicate the presence of tuberculous pleuritis., (© 2011 The Authors. Respirology © 2011 Asian Pacific Society of Respirology.)

Human neutrophil elastase (HNE) and cathepsin G (CatG) are involved in the pathogenesis of a number of inflammatory disorders. These serine proteinases are released by neutrophils and monocytes in case of infection. Wound infection is a severe complication regarding wound healing causing diagnostic and therapeutic problems. In this study we have shown the potential of HNE and CatG to be used as markers for early detection of infection. Significant differences in HNE and CatG levels in infected and non-infected wound fluids were observed. Peptide substrates for these two enzymes were successfully immobilised on different surfaces, including collagen, modified collagen, polyamide polyesters and silica gel. HNE and CatG activities were monitored directly in wound fluid via hydrolysis of the chromogenic substrates. Infected wound fluids led to significant higher substrate hydrolysis compared with non-infected ones. These different approaches could be used for the development of devices which are able to detect elevated enzyme activities before manifestation of infection directly on bandages. This would allow a timely intervention by medical doctors thus preventing severe infections., (© 2011 John Wiley & Sons A/S.)

Background: Cell-free DNA is observed to be more in exudative pleural effusions. Based on this fact development of a clinical chemistry test for classification of pleural effusion will require DNA extraction followed by PCR amplification and electrophoresis. These procedures may not be cost effective for the purpose for classification of pleural effusion as already established parameters are popular for the purpose which can be estimated by comparatively low cost colorimetric procedures. Therefore development of a simple colorimetric test for the classification of pleural fluid based on nucleic acid identification test can be attempted. The aim of this work is to develop such colorimetric test for classification of pleural effusion using only pleural fluid sample., Methods: Cell pellet is obtained from 5 ml pleural fluid which is lysed and subjected to DNA extraction, followed by identification under UV-transilluminator after electrophoresis and orcinol and diphenylamine reaction., Result: Exudates show extractable DNA from 5 ml biofluid (n=52) which are not observed from transudate (n=32). Orcinol reaction is significantly positive in exudates (n=52) compared to the transudates (n=32). Diphenylamine test cannot differentiate exudate from transudate., Conclusion: Orcinol reaction of cell lysate obtained from pleural fluid can classify pleural fluid sample into exudate or transudate., (Copyright 2010 Elsevier B.V. All rights reserved.)

The reactivity of oxide-dependent and nitroxide-forming enzymatic systems of macrophages infected by bacteria, characterized by intraphagosomal (Staphylococcus aureus) and intracytoplasmatic (Listeria monocytogenes) localization in the phagocyte has been investigated. The correlation analysis revealed links between indices of the activity of oxide-dependent and nitroxide-forming enzymatic systems of infected macrophages.

Nitrogen is one of the crucial elements that regulate plant growth and development. It is well-established that plants can acquire nitrogen from soil in the form of low-molecular-mass compounds, namely nitrate and ammonium, but also as amino acids. Nevertheless, nitrogen in the soil occurs mainly as proteins or proteins complexed with other organic compounds. Proteins are believed not to be available to plants. However, there is increasing evidence to suggest that plants can actively participate in proteolysis by exudation of proteases by roots and can obtain nitrogen from digested proteins. To gain insight into the process of organic nitrogen acquisition from proteins by leek roots (Allium porrum L. cv. Bartek), casein, bovine serum albumin and oxidized B-chain of insulin were used; their degradation products, after exposure to plant culture medium, were studied using liquid chromatography-mass spectrometry (LC-MS). Casein was degraded to a great extent, but the level of degradation of bovine serum albumin and the B-chain of insulin was lower. Proteases exuded by roots cleaved proteins, releasing low-molecular-mass peptides that can be taken up by roots. Various peptide fragments produced by digestion of the oxidized B-chain of insulin suggested that endopeptidase, but also exopeptidase activity was present. After identification, proteases were similar to cysteine protease from Arabidopsis thaliana. In conclusion, proteases exuded by roots may have great potential in the plant nitrogen nutrition.

Inflammatory cytokines are reportedly involved in the pathogenesis of chronic subdural hematomas (CSH), and the angiogenesis of hematomas has particularly been in focus. Cyclooxygenase-2 (COX-2) is an essential enzyme for the synthesis of prostaglandin E2 (PGE2). The COX-2-PGE2 pathway has been shown to influence angiogenic factors such as vascular endothelial growth factor (VEGF). We investigated the association of COX-2 expression in the dura mater and outer membrane with the pathogenesis of CSH, and suggested a treatment strategy on the basis of this association. Hematoma fluid and serum samples obtained from 37 patients, and samples of the dura mater and outer CSH membrane obtained from 13 patients during the operation were examined in this study. The concentrations of PGE2 in relation to COX-2 in the hematoma fluid were significantly higher than those in the serum. Immunohistochemical analyses revealed COX-2-positive cells in the outer membrane of CSHs. There was a linear and significant relationship between PGE2 concentration in hematoma fluid and the interval from trauma to initial surgery. COX-2 may play a crucial role during the development of CSHs. Our study might lead to the development of anti-COX-2 treatment options that aim to minimize repeat surgery and choose medical therapy by reducing CSH morbidity and recurrence rate in patients with CSH.

Tuberculous pleural effusion is one of the most common forms of extrapulmonary tuberculosis (TB). The immediate cause of the effusion is a delayed hypersensitivity response to mycobacterial antigens in the pleural space. For this reason microbiological analyses are often negative and limited by the lengthy delay in obtaining results. In areas with high TB prevalence, pleural fluid adenosine deaminase (ADA) levels greater than 40 U/l argue strongly for TB; in contrast, low levels of pleural ADA have high negative predictive value in low-prevalence countries. The specificity of this enzyme increases if only lymphocytic exudates are considered. The shortcoming of the ADA test is its inability to provide culture and drug sensitivity information, which is paramount in countries with a high degree of resistance to anti-TB drugs. Sputum induction (in addition to pleural fluid) for acid-fast bacilli and culture is a recommended procedure in all patients with TB pleurisy. The microscopic-observation drug-susceptibility assay performed on pleural fluid or pleural tissue increases by two to three times the detection of TB over conventional cultures, and it allows for the identification of multidrug-resistant TB. A reasonable management strategy for pleural TB would be to initiate a four-drug regimen and perform a therapeutic thoracentesis in patients with large, symptomatic effusions.

The engraftment of a skin sheet together with transplantation of dermal equivalent was studied on rats. The skin sheet was taken as a source of epithelization material in unhealing wound. The process of wound healing was evaluated by changes of matrix metalloproteinases (MMP) activity levels in wound fluid. It was shown that results of skin sheet transplantation could be predicted by monitoring of wound fluid MMP-2 and MMP-9 activity levels. It was determined that skin sheet engraftment appeared at medium values of wound fluid MMP-2 and MMP-9, and transplanted skin sheet was lysed at high and low values.

Background: The presence of lipocalin-like prostaglandin D synthase (L-PGDS), a dominantly brain derived protein in the cerebrospinal fluid (CSF) with a low concentration in serum, in the subretinal fluid (SRF) of detached retinas, has not been reported. L-PGDS has been demonstrated in the ciliary body and the retinal pigment epithelium in rats., Methods: SRF was sampled during surgery (scleral buckling) of rhegmatogenous retinal detachments in 20 eyes. Biochemical and nephelometric analysis for L-PGDS concentration were performed. Albumin concentrations were determined in the same samples., Results: L-PGDS concentrations in SRF (mean 18.9 mg/L +/- 14.8 mg/L) were markedly higher than the normal L-PGDS concentration in serum (<0.55 mg/L) and appears to decrease with the duration of the retinal detachment (P = 0.0064)., Conclusions: As the subretinal space is not accessible in attached retinas there are no data on normal levels of L-PGDS in the subretinal space. The L-PGDS concentrations measured in the SRF of detached retinas are on average 34 times higher than normal L-PGDS serum concentration and about in the range of normal L-PGDS CSF concentration (15.3 mg/L). As the concentrations of L-PGDS exceed normal serum levels, a choriocapillary transudation is highly improbable as the source of L-PGDS in SRF of detached retinas.

Oxidised regenerated cellulose/collagen matrix (ORC/collagen matrix) modifies wound microenvironments by binding and inactivating excess levels of proteases such as elastase, plasmin and gelatinases in wound exudates. To compare levels of the gelatinases matrix metalloproteinase 2 (MMP-2), elastase and plasmin in wound exudates collected from chronic venous insufficiency patients with venous leg ulcers treated with either an ORC/collagen matrix or a standard control therapy. During a 12-week treatment period, wound exudate samples were obtained from a control group of 10 patients treated with a hydrocolloid dressing and a treatment group of 17 patients treated with a combination of ORC/collagen matrix and hydrocolloid dressing. On admission and days 5, 14 and every subsequent 14th day, ulcers were photographed to determine healing rate and changes in ulcer appearance, and MMP-2 concentration and the gelatinase, elastase and plasmin activities were analysed from wound exudates. The patients treated with ORC/collagen matrix showed a significant decrease in elastase, plasmin and gelastinase activity as compared with the control group, with no significant difference in the MMP-2 concentrations between the two groups. The results show a significant and immediate reduction in protease activity in wound exudates from venous leg ulcers treated with ORC/collagen.

Background: The pathology of chronic wounds is often characterized by elevated levels of proinflammatory cytokines [e.g. tumour necrosis factor (TNF)-alpha and interleukin (IL)-1beta], proteases [e.g. matrix metalloproteinases (MMPs)] and neutrophil elastase. MMPs specifically have been implicated by a number of studies as the major protease family responsible for the degradation of key factors critical to the ulcer's ability to heal., Objectives: To assess individual MMPs in chronic wound fluid (CWF) in order to develop improved treatments for chronic ulcers., Methods: Collagen type I and IV zymography, immunoprecipitation followed by a substrate activity assay, and an indirect enzyme-linked immunosorbent assay were all used to analyse MMP levels in CWF., Results: Our studies demonstrate that there is excessive protease activity in CWF compared with both human serum and acute wound fluid (AWF), which can be specifically attributed to MMPs as determined through a MMP-inhibitor study. Multiple MMPs were immunoprecipitated from the CWF samples and MMP-9 was identified as the predominant protease in CWF, with significantly elevated activity levels in CWF compared with AWF. In addition, the clinical status of the ulcer is directly associated with the amounts of MMP-9 present in the wound fluid. Therefore, this study suggests that higher levels of MMP-9 in chronic wound fluid correlate with a clinically worse wound., Conclusions: In view of these results, it is hypothesized that a specific inhibitor of MMP-9 could potentially be more therapeutically effective than general MMP inhibitors in modulating chronic ulcers towards a healing state.

One of the problems in wound treatment optimization is the necessity of an effective and objective method of laboratory wound process monitoring. In present study the current wound process was estimated by changes in MMP-2 and MMP-9 activities in the wound fluid. An original model was used in this work to study correlation of morphological structure of the wound with the activities of MMP-2 and MMP-9 in wound fluid at various types of wound process. Protein fractions of the coelomic liquid from regenerating sea star Asterias rubens were used as the substances changing the wound process. The correlation of wound process with MMP-2 and MMP-9 activities in wound fluid was revealed on the basis of correlation analysis.

Background: We investigated the activity of matrix metalloproteinase (MMP)-2 and -9 and the protein levels of MMP-1, -3, -8 and the tissue inhibitor of MMPs (TIMP)-1 in the subretinal fluid (SRF) of patients with rhegmatogenous retinal detachment (RRD) and establishment of potential correlations with clinical parameters., Methods: Thirty-seven SRF from RRD patients and nine vitreous samples from the human eye of organ donors (controls) were collected and assayed for MMP-1,-3,-8 and TIMP-1 levels using ELISA and for MMP-2 and -9 activity employing gelatin zymography., Results: MMP-1, MMP-3, MMP-8, proMMP-2, proMMP-9, MMP-9 and TIMP-1 levels were higher in SRF compared with vitreous fluid. Overall, MMPs and TIMPs were differentially expressed in SRF with respect to duration and extent of RRD, as well as to stage of proliferative vitreoretinopathy. Regression analysis for all data indicated that a model consisting of MMP-3, MMP-8 and proMMP-9 could estimate the extent of RRD., Conclusions: MMPs and TIMP-1 levels are elevated in SRF during RRD. A regression model consisting of MMP-3, MMP-8 and proMMP-9 may be proved to be of potential use in providing information for evaluation of the extent of RRD.

Objective: Heparanase, an endoglycosidase that cleaves heparan sulphate, is frequently expressed in carcinomas and was suggested to play a role in cell invasion and metastasis. We investigated whether heparanase expression may serve as a reliable marker to discriminate benign mesothelial cells from malignant cells shed into body cavities., Methods and Results: Cytological smears of effusions from 51 hospitalized patients were immunostained for heparanase. Strong immunoreactivity was noted in 35 of 40 (88%) carcinoma samples and in all three malignant mesothelioma cases. Only rare (<3%) reactive mesothelial cells were noted showing a faint negligible staining. Specificity was 100%, sensitivity 88%, and positive and negative predictive values were 100% and 89% respectively., Conclusions: Our results suggest that heparanase may be of value as a complementary component in a diagnostic panel of markers, contributing to its reliability and accuracy.

Venous ulcer fibroblasts have been demonstrated to have low growth rates in response to platelet-derived growth factor (PDGF). Mitogen-activated protein kinase (MAPK) is an important signal transduction mechanism that regulates growth, differentiation, and apoptosis in eukaryotic cells. PDGF binds PDGF receptors that activate a multitiered signaling cascade involving MAPK. We hypothesize that the growth regulation in venous ulcer fibroblasts is dependent on the MAPK extracellular signal-regulated kinase (ERK) pathway in the presence of PDGF. Fibroblasts (fb) were isolated from 8 patients with venous ulcers (w-fb) and the normal skin (n-fb) of the ipsilateral thigh via punch biopsies. Fb were plated at 1,500 cells/dish and treated with PDGF-AB (10 ng/mL) for 15 days. Growth rates were determined. Immunoblot analysis of MAPK ERK for n-fb and w-fb were analyzed. To determine if PDGF-stimulated w-fb and n-fb utilized the MAPK ERK pathway in a dependent manner, the upstream kinase MAPK kinase 1 (MEK 1) was inhibited by PD 98059. In addition, fb were treated with chronic venous ulcer wound fluid (WF) to study its effect on MAPK ERK. In the presence of PDGF, growth rates were substantially lower in w-fb than in n-fb, and MAPK was activated in 6/8 w-fb and in only 2/8 n-fb. Fibroblasts expressing MAPK had significantly reduced cell proliferation compared to fibroblasts not expressing MAPK (p = 0.023). PD 98059 significantly inhibited w-fb and n-fb cell proliferation from basal level, which was reversible with addition of PDGF. In neonatal fibroblasts WF demonstrated inhibition of MAPK ERK over time and addition of PD98059 was not additive. This study suggests that the MAPK ERK pathway is important for cell proliferation in venous ulcer fibroblasts. In the presence of PDGF, fibroblasts with decreased growth rate express MAPK, and proliferation is further abrogated with addition of MEK 1 inhibitor, suggesting the importance of the MAPK ERK pathway regulating w-fb and n-fb proliferation. Although the majority of w-fb activated the MAPK ERK pathway in the presence of PDGF, proliferation was significantly attenuated, indicating that other MAPK inhibitory pathways are competing. Venous ulcer wound fluid directly inhibits the MAPK ERK pathway, suggesting that the venous ulcer wound environment has negative trophic factors that effect fibroblasts proliferation and ulcer healing.

Measurement of pleural adenosine deaminase activity (ADA) is a useful diagnostic tool for tuberculous pleurisy, but false-positive findings from non-tuberculous effusions have been reported. In order to improve diagnostic value of ADA it is recommended to estimate activity of both ADA1 and ADA2 izoenzymes or 2'-deoxyadenosine/adenosine activity ratio. In order to evaluate ADA as a diagnostic parameter total ADA, with adenosine as a substrate, and 2'-deoxyadenosine/adenosine activity ratio were measured in tuberculous and malignant pleural effusions. Altogether, 26 pleural exudates (11 tuberculous and 15 malignant) were selected. ADA either with adenosine or 2'-deoxyadenosine was determined by colorimetric method of Giusti. Each pleural fluid sample was diluted prior to the assay (1:8) to avoid enzyme inhibition which was observed in nondiluted pleural effusions. The ADA level reached the diagnostic cut-off set for tuberculous effusions (40 U/L) in every 11 tuberculous exudates with the mean value of 85,3+/-47,1 U/L; in 9 of these the 2'-deoxyadenosine/adenosine ratio was less than 0,45. In the malignant group of patients, no one ADA level exceed 40 U/L, being estimated at 10,6+/-7,7 U/L (p<0,001). In 10 of these 15 exudates the 2'-deoxyadenosine/adenosine ratio was undetectable, in four it was less than 0,45 and only in one it was over 0,45. We concluded that ADA measured by the Giusti method proceeded by the dilution 1:8 of the pleural effusion samples very good differentiates tuberculous from malignant pleurisy, without the necessity to determine the 2'-deoxyadenosine/adenosine ratio. The investigation needs to be continued on the more numerous groups of patients.

Objective: The purpose of this study was to evaluate the usefulness of a new parameter, pleural adenosine deaminase (PADA), for separating transudative pleural effusion from exudative pleural effusion, and to compare the results with other tests (albumin gradient and protein gradient)., Methods: From November 2001 to January 2003, 359 consecutive patients with pleural effusion who underwent a diagnostic thoracentesis were included in the study. Effusions were individually classified as transudates or exudates after the careful evaluation of all clinical data and biochemical parameters of pleural fluid and serum of patients on the basis of Light's criteria. The means and standard deviations of PADA, pleural/serum ADA (P/S ADA) ratio, albumin gradient and protein gradient were evaluated for transudative and exudative effusions. The best cut-off values for each test were identified by using the receiver operating characteristic (ROC) curve. The optimum cut-off level was determined by selecting points of test values that provided the greatest sum of sensitivity and specificity., Results: There were 113 transudates and 246 exudates. For each test, differences in mean value between the transudate group and the exudate group were statistically significant (t test, P<0.001). The optimum cut-off levels for PADA and P/S ADA were 15.3 U/L and 0.66 U/L, respectively. ROC analysis confirmed previous recommendations for albumin gradient (12 g/L) and protein gradient (31 g/L). For detecting exudates, the PADA test yielded a sensitivity and specificity of 85.8% and 82.3%, respectively. Sensitivity and specificity of the albumin gradient were found to be 88.5% and 79.3%, and of the protein gradient 85% and 83.2%, respectively. The areas under the curve (AUC) data and accuracy demonstrated similar discriminative properties in the examined tests., Conclusions: The measurement of PADA is suggested as a reliable test in the separation of pleural exudates from transudates with accuracy similar to that of the albumin gradient and protein gradient.

The aim of this study was to determine neutrophil elastase levels (NE) in periapical exudates and to evaluate its relationship with clinical signs and symptoms of endodontically involved teeth. A total of 42 teeth with periapical periodontitis of 37 patients were clinically examined and spontaneous pain, swelling, pain on palpation or percussion, sinus tract formation and pus discharge were recorded. Additionally, periapical lesion size was measured on periapical radiographs. Periapical exudate samples were obtained during routine root canal treatment by using paper points. Enzyme levels were determined by spectrophotometric assays using the NE specific substrate N-methoxysuccinil-Ala-Ala-Pro-Val-p-nitroanilide. The teeth with signs and symptoms showed higher NE levels than the teeth without symptoms (p<0.05). The differences were significant when NE levels were presented as total amounts instead of concentration. This study demonstrated that NE in periapical lesions related with clinical symptoms and total enzyme amount may be more reliable mode of data presentation.

Orchestration of the rapid formation and reorganization of new tissue observed in wound healing involves not only cells and polypeptides but also the extracellular matrix (ECM) microenvironment. The ability of heparan sulfate (HS) to interact with major components of the ECM suggests a key role for HS in maintaining the structural integrity of the ECM. Heparanase, an endoglycosidase-degrading HS in the ECM and cell surface, is involved in the enzymatic machinery that enables cellular invasion and release of HS-bound polypeptides residing in the ECM. Bioavailabilty and activation of multitude mediators capable of promoting cell migration, proliferation, and neovascularization are of particular importance in the complex setting of wound healing. We provide evidence that heparanase is normally expressed in skin and in the wound granulation tissue. Heparanase stimulated keratinocyte cell migration and wound closure in vitro. Topical application of recombinant heparanase significantly accelerated wound healing in a flap/punch model and markedly improved flap survival. These heparanase effects were associated with enhanced wound epithelialization and blood vessel maturation. Similarly, a marked elevation in wound angiogenesis, evaluated by MRI analysis and histological analyses, was observed in heparanase-overexpressing transgenic mice. This effect was blocked by a novel, newly developed, heparanase-inhibiting glycol-split fragment of heparin. These results clearly indicate that elevation of heparanase levels in healing wounds markedly accelerates tissue repair and skin survival that are mediated primarily by an enhanced angiogenic response.-Zcharia, E., Zilka, R., Yaar, A., Yacoby-Zeevi, O., Zetser, A., Metzger, S., Sarid, R., Naggi, A., Casu, B., Ilan, N., Vlodavsky, I., Abramovitch, R. Heparanase accelerates wound angiogenesis and wound healing in mouse and rat models.

Purpose: The purpose of this study was to quantify the expression changes of gelatinase-A and -B (matrix metalloproteinase [MMP]-2 and MMP-9) in a series of chondral, meniscal, and synovial cultures of knee osteoarthritis (OA) for investigation of the possible roles of the cartilage, menisci, and synovia and the efficacy of arthroscopic debridement., Type of Study: A biochemical study., Methods: In 43 consecutive patients with knee OA undergoing arthroscopic debridement, we examined the amount of MMP-2 and MMP-9 in a series of chondral, meniscal, and synovial cultures. We also compared the gene expressions of MMP-2 and MMP-9 and membrane-type 1 MMP (MT1-MMP) in the chondral, meniscal, and synovial cultures using reverse transcription polymerase chain reaction (RT-PCR)., Results: Latent and activated forms of MMP-2 were produced in all series of chondral, meniscal, and synovial cultures, and the levels in lesional cultures were significantly higher than those in paralesional ones (P <.001). Moreover, the latent form of MMP-9 (proMMP-9) appeared in 29 of 37 series of synovial cultures and in 13 of 40 series of meniscal cultures. In meniscal cultures after 24 hours of incubation and synovial cultures after 3 and 24 hours of incubation, the level of proMMP-9 in lesional cultures was significantly higher than that in paralesional ones (P <.001). The activated form of MMP-9 appeared in 10 of 37 series of synovial cultures, and the level in lesional cultures was significantly higher than that in paralesional ones (P <.05). Furthermore, MMP-2, -9, and MT1-MMP mRNA levels of lesional areas also showed the increased expression in RT-PCR., Conclusions: Our data confirm that tissue repair of OA is ascribable to enzymic digestion of the extracellular matrix ex vivo. When technically appropriate, arthroscopic debridement for the pathologic lesions of OA, such as meniscal tears, chondral lesions, and hypertrophic villi, may be beneficial to the process of early cases. Still, it should be carefully studied for its overall effect and mechanism in vivo., Clinical Relevance: The effectiveness of arthroscopic debridement for the treatment of knee OA is controversial. The present study provides the possible mechanism of the positive effects of arthroscopic debridement in basic science.

The objective was to study the expression of inducible nitric oxide synthase (NOS II) in and NO production by human blood neutrophils and in in vivo exudated neutrophils. Cellular expression of NOS II was evaluated by flow cytometry in whole blood, in isolated blood neutrophils, and in neutrophils obtained by exudation in vivo into skin chambers. Neutrophil NOS II was also demonstrated by Western blotting. Uptake of 3H-labelled L-arginine was studied in vitro and NOS activity measured in a whole cell assay by the conversion of 3H-arginine to 3H-citrulline. In contrast to unseparated blood cells, NOS II was demonstrable both in isolated blood neutrophils and exudated cells. The failure to detect NOS II by flow cytometry in whole blood cells thus proved to be due to the quenching effect of hemoglobin. Western blotting revealed a 130 kD band corresponding to NOS II in isolated blood neutrophils, but detection was dependent on diisopropylfluorophosphate for proteinase inhibition. L-arginine was taken up by neutrophils, but enzymatic activity could not be demonstrated. We conclude that human neutrophils constitutively express NOS II, but that its demonstration by FITC-labelling is inhibited by hemoglobin-mediated quenching in whole blood samples.

We conducted a comparative study of the protective effects of chitin, chitosan, and N-acetyl chitohexaose (NACOS-6) against mice infected intravenously or intraperitoneally with Pseudomonas aeruginosa and Listeria monocytogenes. Mice pretreated with chitin, chitosan, and NACOS-6 showed resistance to intraperitoneal infections by both microbes. Only mice pretreated with chitin and chitosan showed resistance to intravenous infections by both microbes. The number, active oxygen generation, and myeloperoxidase activity of peritoneal exudate cells (PEC) in the chitin, chitosan, and NACOS-6-treated mice were greater than those of the untreated mice. Also, these PEC factors from mice pretreated with chitin and chitosan were greater than those from the NACOS-6-treated mice.

The influence of burn fluid and its matrix metalloproteinases (MMPs) on Balb/3T3 cells was studied. The influence of burn fluid was assessed by morphology and specific functional activities of cells characteristic of the healing process--proliferation, monolayer contraction and migration of cells in wound model. The presence of burn fluid in cultivating medium accelerated cell proliferation by 2.5 times compared to normal conditions, promoted fibroblast monolayer contraction, and accelerated cell migration on the wound surface, thus stimulating cell functions necessary for successful heating. This effect is partly due to MMPs. The burn fluid contains, presumably, some additional factors not inhibited by specific MMP inhibitors EDTA and 1,10-phenantrolin. These factors may stimulate migration and proliferation of cells. The presence of 1-2% burn fluid is sufficient for enhancing cell proliferation.

The dynamics of matrix metalloproteinases (MMP), as well as of fibronectin concentration in wound and burn fluids was traced. The wound fluid proteolytic activity was studied by gelatin zymography method. The data on degradation of fibronectin and various laminin isoforms by wound fluid proteases show that laminin-1, laminin-2/4 and fibronectin were degraded by wound fluid into small fragments. Remodelling of extracellular matrix proteins occurs. Dynamics of MMP-2 and MMP-9 content in wound or burn fluids as well as that of adhesive protein fibronectin content could be used as a base for development of method of controlling the extracellular matrix remodelling process.