Assessment of serum and synovial fluid MMP-3 and MPO as biomarkers for psoriatic arthritis and their relation to disease activity indices.

Research on biomarkers for the diagnosis and monitoring of psoriatic arthritis (PsA) is ongoing. The purpose of this study was to assess the potential of serum and synovial fluid matrix metalloproteinase-3 (MMP-3) and myeloperoxidase (MPO) as biomarkers for PsA and their relation to disease activity indices. This case-control study involved 156 psoriatic arthritis patients, 50 gonarthrosis patients, and 30 healthy controls. The target parameters were measured with the enzyme-linked immunosorbent assay (ELISA) kits. Serum MMP-3 and MPO levels were elevated in the PsA patients in comparison to the two control groups (p < 0.001) and distinguished PsA from GoA patients and healthy controls with 100% accuracy. Synovial MMP-3 discriminated PsA from GoA patients irrespective of the presence of crystals (AUC = 1.00). PsA patients with crystals in the synovial fluid had elevated synovial MPO (p < 0.001) and were distinguished from PsA patients without crystals with accuracy of 88.50% and from GoA patients with accuracy of 88.30%. Synovial fluid MPO was positively associated with the following indicators of disease activity: VAS (rs = 0.396); DAPSA (rs = 0.365); mCPDAI (rs = 0.323). Synovial MMP-3 showed a weaker positive association with DAPSA (rs = 0.202) and mCPDAI (rs = 0.223). Our results suggest that serum MMP-3 and MPO could serve as biomarkers for PsA. Synovial fluid MMP-3 showed a potential as a biomarker for PsA versus GoA. Synovial MPO could be utilized as a marker for the presence of crystals in PsA patients.

Target-Specific Fluorescence-Mediated Tomography for Non-Invasive and Dynamic Assessment of Early Neutrophil Infiltration in Murine Experimental Colitis.

The role of neutrophils in the pathogenesis of inflammatory bowel disease (IBD) is still only incompletely understood. Here, we evaluated target-specific fluorescence-mediated tomography (FMT) for visualization of neutrophil infiltration in murine experimental DSS-induced colitis. Colitis was assessed using clinical, endoscopic, and histopathological parameters. Intestinal neutrophil infiltration was determined at day 0, 4, and 10 by targeted FMT after injection of a neutrophil-specific fluorescence-labelled monoclonal antibody (Gr-1). Complementary, immunofluorescence tissue sections with Gr-1 and ELISA-based assessment of tissue myeloperoxidase (MPO) served as the gold standard for the quantification of neutrophil infiltration. Colitic animals showed decreasing body weight, presence of fecal occult blood, and endoscopic signs of inflammation. FMT revealed a significantly increased level of fluorescence only four days after colitis induction as compared to pre-experimental conditions (pmol tracer 73.2 ± 18.1 versus 738.6 ± 80.7; p < 0.05), while neither body weight nor endoscopic assessment showed significant changes at this early time. Confirmatory, post-mortem immunofluorescence studies and measurements of tissue MPO confirmed the presence of increased neutrophil infiltration in colitic mice compared to controls. Concluding, Gr-1 targeted FMT can detect early colonic infiltration of neutrophils in experimental colitis even before clinical symptoms or endoscopic alterations occur. Therefore, FMT might be an important tool for repetitive and non-invasive monitoring of inflammatory cell infiltrate in intestinal inflammation.

Assessment of Myeloperoxidase and Nitric Levels around Dental Implants and Natural Teeth as a Marker of Inflammation: A Comparative Study.

INTRODUCTION: Dental implants form the mainstay of dental treatment involving rehabilitation of missing teeth. One of the major concerns for the clinicians doing dental implants is the postsurgical failure of dental implants. Success of dental implants is dependent upon the skills of the surgeon and the amount and quality of the bone remaining at the edentulous area where dental implant has to be placed. Myeloperoxidase (MPO) and nitrites are few of the enzymes and molecules which are said to be altered in inflammation. However, their exact role in the inflammatory processes around natural tooth and dental implant is still unclear. Hence we comparatively evaluated the levels of MPO and nitrites in the areas around the dental implants and natural teeth. MATERIALS AND METHODS: The present study comprises 42 patients who underwent prosthetic rehabilitation by dental implants from 2011 to 2014. Depth of probing value (DP), score of plaque index (SPI), gingival index (GI), and index of gingival bleeding time (GBT) were evaluated for the assessment of the periimplant soft tissue changes. Assessment of inflammation around the dental implant surface and around natural tooth was done based on the readings of these parameters. For the measurement of the MPO levels, spectrophotometric MPO assay was used. All the results were analyzed by Statistical Package for the Social Sciences (SPSS) software. RESULTS: The mean plaque index values were 1.56 and 0.97 in periodontitis cases of natural teeth and inflamed cases of dental implants respectively. While comparing mean plaque index, mean probing depth, and mean gingival bleeding index in between the two groups, significant difference was obtained. Mean MPO concentration in periodontitis and gingivitis cases in natural teeth were 0.683 and 0.875 U/µL, while in inflamed dental implant cases, the mean value was 0.622 U/µL. While comparing the total MPO levels, total nitrite levels, and total nitrite concentration in between two study groups, significant difference was obtained. On comparing the healthy and periodontitis cases in natural teeth, significant difference was obtained. CONCLUSION: In the inflammatory processes occurring around dental implant and natural teeth, MPO and NO make some amount of significant contribution. CLINICAL SIGNIFICANCE: The present study enforces on the role of MPO and nitrite as diagnostic and prognostic marker.

Toxicity assessment of simulated urban runoff containing polycyclic musks and cadmium in Carassius auratus using oxidative stress biomarkers.

The objective of this study was to assess potential toxic effects of simulated urban runoff on Carassius auratus using oxidative stress biomarkers. The activity of antioxidant enzymes including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and the content of malondialdehyde (MDA) in the liver of C. auratus were analyzed after a 7-, 14- and 21-day exposure to simulated urban runoff containing galaxolide (HHCB) and cadmium (Cd). The results showed that the activity of antioxidant enzymes and the content of MDA increased significantly exposed to the simulated urban runoff containing HHCB alone or mixture of HHCB and Cd. The activity of the investigated enzymes and the content of MDA then returned to the blank level over a longer period of exposure. The oxidative stress could be obviously caused in the liver of C. auratus under the experimental conditions. This could provide useful information for toxic risk assessment of urban runoff.

Regulação cruzada entre peroxidases e indolamina 2.3 dioxigenase no controle da metabolização do triptofano / Peroxidases and indoleamine 2.3 dioxygenase crosstalk modulating tryptophan metabolization

Triptofano (TRP) é metabolizado por duas vias, a via serotonérgica e a via das quinureninas. Na via serotonérgica, TRP é metabolizado a serotonina (5-HT) e, em algumas células, à melatonina (MLT) que pode ser oxidada à N1-acetil-N2-formil-5- metoxiquinuramina (AFMK) e N1-acetil-5-metoxiquinuramina (AMK) por ação de peroxidases. Na via das quinureninas o TRP é diretamente metabolizado à N formilquinurenina (NFK) e em seguida a quinurenina (QUIN). A enzima indolamina 2, 3 dioxigenase (IDO) é uma das responsáveis por esta reação. Dada a importância da IDO na tolerância imunológica e pelo fato desta enzima ser induzível nos propusemos a avaliar a existência de uma regulação cruzada entre esta enzima e a via serotonérgica. Avaliando a interferência de AMK sobre a ação de IDO e a interferência de QUIN sobre a formação de AFMK por peroxidases, observamos uma possível interação entre as vias. AMK é um inibidor competitivo clássico de IDO e o Ki encontrado foi de 0,98 mM. QUIN é um inibidor acompetitivo linear simples da formação de AFMK e o Ki encontrado foi de 0,1 mM. A inibição da formação de AFMK também ocorre para a peroxidase humana (mieloperoxidase, MPO). Além de representarem uma regulação cruzada utilizada in vivo, as inibições encontradas podem ser relevantes para a proposta de novos inibidores de IDO e MPO na terapia imunomodulatória. Dado o nosso interesse pelas enzimas IDO e MPO, avaliamos ainda a localização intracelular destas enzimas em células de peritônio de camundongo, tanto residente como ativada com concanavalina A (Con A). O estímulo com Con A representa uma ativação de linfócitos T mediado por interferon gama (IFN-γ) e foi usado como modelo experimental para avaliar condições de localização em células ativadas. Por imunocitoquímica verificamos que IDO e MPO localizam-se próxima à membrana plasmática sendo que uma leve dispersão apenas de MPO foi observada em células ativadas com Con A. A localização intracelular das duas enzimas é no...

Tryptophan (TRP) is metabolized by two mains pathways, the serotoninergic pathway and the kynurenine pathway. In the serotoninergic pathway, TRP is metabolized to serotonin (5-HT) and, in some cells, to melatonin (MLT). The later can even be oxidized to acetyl-N1-N2-formyl-5-methoxykynuramine (AFMK) and N1-acetyl-5 -methoxykynuramine (AMK) by peroxidases. In the kynurenine pathway, TRP is metabolized to N-formylkynurenine (NFK) and to kynurenine (KYN). Indoleamine 2, 3 dioxygenase (IDO) is one of those responsible for this reaction. Since IDO is importat in immune tolerance and the fact that this enzyme is inducible by cytokines we proposed whether there is a cross regulation between this enzyme and the serotoninergic pathway. A possible interaction between MLT and TRP oxidation pathways was shown by the AMK influence on IDO activity and QUIN interference on AFMK formation by peroxidases. AMK was shown to be an IDO classical competitive inhibitor with a Ki of 0.98 mM. QUIN was a peroxidase (horseradish peroxidase, HRP) classical uncompetitive inhibitor and Ki was found to be 0,1 mM. AFMK formation inhibition was also found in human peroxidase (myeloperoxidase, MPO). Beyond the in vivo crosstalk, new IDO and MPO inhibitors in immunomodulatory therapy would be proposed by the compounds shown in this study. Given our interest in IDO and MPO, we also evaluated their intracellular localization in both resident and concanavalin A (Con A) activated mice peritoneum cells. Con A stimulation is a IFN-γ mediated T lymphocytes activation and was our experimental model to evaluate activated cells. In light microscopy we observed IDO and MPO localization near the membrane and MPO only had a dispersed localization in Con A activated cells. Cytoplasm, nucleus and vesicles were the intracellular localization of both enzymes. Interestingly, we found MPO in isolated cells and in cell clusters of two or more cells. MPO was founded on macrophages, B1 cells and cell clusters by...

Iridium oxide nanomonitors: clinical diagnostic devices for health monitoring systems.

The objective of this research is to demonstrate the potential of iridium oxide (IrOx) nanowires based device towards detection of proteins that are disease biomarkers. This device is based on electrical detection of protein biomarkers wherein an immunoassay is built onto the iridium oxide nanowires that in turn undergoes specific electrical parameter perturbations during each binding event associated with the immunoassay. Detection of two inflammatory proteins C-reactive protein (CRP) and Myeloperoxidase (MPO) that are biomarkers of cardiovascular diseases is demonstrated. The performance metrics of the device in response to the two biomarkers in pure form and in serum samples were evaluated and compared to standard ELISA assays. The methodology that has been adopted is based on measuring impedance and calibrating its change in magnitude with concentration of proteins. We demonstrate the following performance metrics: limits of detection up to 1 ng/ml for CRP and 500 pg/ml for MPO in pure and serum samples; linear dynamic range of detection from 10 ng/ml to 100 microg/ml for CRP and 1 ng/ml to 1 microg/ml for MPO and cross-reactivity contained at less than 10% of selective binding for both the inflammatory proteins. Iridium oxide has an ability to detect very small changes to the surface charge and this capability is utilized for achieving the performance metrics and forms the basis of the key innovations of this technology, which are, improving the selectivity and sensitivity of detection.

Photosynthesis and oxidative stress in the restinga plant species Eugenia uniflora L. exposed to simulated acid rain and iron ore dust deposition: potential use in environmental risk assessment.

The Brazilian sandy coastal plain named restinga is frequently subjected to particulate and gaseous emissions from iron ore factories. These gases may come into contact with atmospheric moisture and produce acid rain. The effects of the acid rain on vegetation, combined with iron excess in the soil, can lead to the disappearance of sensitive species and decrease restinga biodiversity. The effects of iron ore dust deposition and simulated acid rain on photosynthesis and on antioxidant enzymes were investigated in Eugenia uniflora, a representative shrub species of the restinga. This study aimed to determine the possible utility of this species in environmental risk assessment. After the application of iron ore dust as iron solid particulate matter (SPM(Fe)) and simulated acid rain (pH 3.1), the 18-month old plants displayed brown spots and necrosis, typical symptoms of iron toxicity and injuries caused by acid rain, respectively. The acidity of the rain intensified leaf iron accumulation, which reached phytotoxic levels, mainly in plants exposed to iron ore dust. These plants showed the lowest values for net photosynthesis, stomatal conductance, transpiration, chlorophyll a content and electron transport rate through photosystem II (PSII). Catalase and superoxide dismutase activities were decreased by simulated acid rain. Peroxidase activity and membrane injury increased following exposure to acid rain and simultaneous SPM(Fe) application. Eugenia uniflora exhibited impaired photosynthetic and antioxidative metabolism in response to combined iron and acid rain stresses. This species could become a valuable tool in environmental risk assessment in restinga areas near iron ore pelletizing factories. Non-invasive evaluations of visual injuries, photosynthesis and chlorophyll a fluorescence, as well as invasive biochemical analysis could be used as markers.

Fecal lactoferrin, myeloperoxidase and serum C-reactive are effective biomarkers in the assessment of disease activity and severity in patients with idiopathic ulcerative colitis.

BACKGROUND AND AIM: Disease activity and severity of ulcerative colitis (UC) is assessed using colonoscopy, which is invasive, costly and has poor patient acceptability. The role of non-invasive biomarkers of intestinal inflammation in the evaluation of patients with UC is not known. The aim of the study was to examine the role of serum C-reactive protein (SCRP), fecal myeloperoxidase (FMPO) and fecal lactoferrin (FLF) in assessing disease severity, activity and response to therapy. METHODS: Consecutive patients with idiopathic UC (IUC) attending our hospital from July 2005 to September 2006 were studied. All underwent clinical, endoscopic and histological assessment for disease activity, extent, severity and estimation of SCRP, FMPO and FLF levels at baseline and follow up (FU). An equal number of healthy age-matched controls were studied for biomarker levels. RESULTS: A total of 37 patients (mean age 37 +/- 12 years) were studied. All three biomarkers were elevated more often in the cases than in the controls (all P = 0.000). Cases with severe IUC had higher CRP, MPO and FLF titers than those without severe IUC. At FU, a significant fall in biomarker levels paralleled the reduction in Mayo's scores. All three biomarkers showed a high degree of correlation with each other. The areas under the curve for FLF, MPO and CRP were 1.00, 0.867 and 0.622, respectively. The sensitivity and specificity of markers were: FLF (94%, 100%), FMPO (89%, 51%) and SCRP (24%, 100%). CONCLUSION: Biomarkers are useful in assessing disease activity, severity and response to therapy in patients with UC. They showed a high degree of correlation with each other.

Use of pharmacodynamic endpoints for the evaluation of levofloxacin for the treatment of acute maxillary sinusitis.

Sinusitis remains 1 of the most common reasons for antimicrobial prescriptions in the United States, with health care costs approaching $4 billion annually. We utilized the serial sinus aspirate sampling (SSAS) technique to obtain daily specimens to evaluate the time course of drug effect in patients with acute maxillary sinusitis. Eighteen patients with a radiologically confirmed acute maxillary sinusitis were enrolled into a study evaluating the relationship between levofloxacin exposure and the time course of antimicrobial effect using SSAS. SSAS was performed daily during therapy for bacteriologic evaluation. Six steady-state levofloxacin concentrations were obtained. Levofloxacin plasma and sinus aspirate concentrations were modeled using Monte Carlo Parametric Expectation Maximization algorithm implemented in S-ADAPT 1.53. Endpoints evaluated included time to resolution of signs and symptoms and time to sinus sterilization. Among the 18 enrolled patients, 15 were clinically evaluable. From these, 1 Streptococcus pneumoniae, 3 Haemophilus influenzae, 1 Moraxella catarrhalis, 1 Corynebacterium spp., and 1 coagulase-negative Staphylococcus organisms were isolated, with the latter 2 organisms being likely contaminants. For the pathogens, levofloxacin MIC values ranged from 0.03 to 2 mg/L. All pathogens were eradicated by the 4th day of therapy. The median and mean time to sinus sterilization (pathogens only) was 1 and 1.4 days, respectively. The median time to resolution of each sign and symptom ranged from 1.5 to 12-19 days, with the 83% of total signs and symptoms resolved by the end of therapy (day 5). The mean plasma area under the concentration-time curve (AUC) (mg x h/L) was 100.1 (n = 14, %CV = 27). Plasma AUC/MIC ratios ranged from 33.9 to 1696 for isolated pathogens. In this pilot SSAS study, levofloxacin rapidly eradicated isolated pathogens from the maxillary sinus.

Development and validation of a screening procedure for the assessment of inhibition using a recombinant enzyme.

Myeloperoxidase (MPO, E.C. 1.1.11.7) is a heme-containing enzyme that catalyses the synthesis of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H2O2) and chlorine anions. The production of HOCl is kept under strict control of neutrophils. However, in several pathological conditions, MPO is leaked in the extracellular fluid, which involves an over-production of reactive oxygen species like HOCl and promotes the damages caused by neutrophils. As a consequence, the inhibition of MPO by various agents has been investigated and a variety of molecules have been evaluated for this activity in different models. The present study aims to describe and validate a rapid screening method based on the taurine assay and using a recombinant MPO. After validation of the stock solutions used during the experiments, the amount of MPO for the completion of the reaction was measured and fixed to an optimal value. The inhibiting concentration at 50% of flufenamic acid (taken as a reference molecule) was then assessed in both a simple tube test and a microplate test and delivered similar results (1.3+/-0.2 microM vs 1.4+/-0.2 microM, P=0.2). Finally, different molecules able to inhibit MPO were evaluated in this rapid assay system providing results comparable to literature. The high throughput screening is undoubtedly a first line assessment method which affords the selection of inhibitors and permits to reduce the number of candidates for a further elucidation of the mechanism of MPO inhibition.

Assessment of Schistosoma mansoni induced intestinal inflammation by means of eosinophil cationic protein, eosinophil protein X and myeloperoxidase before and after treatment with praziquantel.

Faecal concentrations of eosinophil cationic protein (ECP), eosinophil protein X (EPX) and myeloperoxidase (MPO) were measured in extracts of stool samples obtained from a cohort of people (n=182) living in Bugoigo, a fishing community on the Eastern shore of Lake Albert, Buliisa District, in North Western Uganda where Schistosoma mansoni is endemic. Samples were collected before treatment and 5, 15, 20 and 52 weeks after treatment with praziquantel. Significantly increased levels of faecal ECP and EPX were found in S. mansoni infected individuals (n=155) compared to the levels found in stools from non-infected (n=27) (median values ECP: 11.3 microg/g vs. 5.9 microg/g, P=0.005, and EPX: 413.5 ng/g vs. 232.2 ng/g, P=0.045). An increased level of MPO was also found among the infected individuals compared to the non-infected 11.6 mu/g vs. 5.3 mu/g, P=0.07). Significant but weak correlations were found between faecal egg counts and faecal concentrations of ECP and EPX. Treatment with praziquantel induced a significant decline in both ECP and EPX, but only a non-significant reduction in faecal MPO. Following reinfection and despite of very low infection intensities, the protein levels increased significantly reaching the pre-treatment level (ECP and EPX) or levels significantly higher than the pre-treatment levels (MPO). This response pattern may imply a rebound effect during reinfection following treatment and resolution of immune regulatory immunosuppressive mechanisms in function during the chronic infection.

Differential effects of macrophage inflammatory chemokine-2 and keratinocyte-derived chemokine on hemorrhage-induced neutrophil priming for lung inflammation: assessment by adoptive cells transfer in mice.

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 +/- 5 mmHg and were then administered anti-MIP- 2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringer's lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; microU/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (increased MPO, increased % esterase+ cells in tissue) into the lung and local tissue inflammation (increased IL-6/MIP-2, decreased IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.

An assessment of the effect of Ginkgo Biloba EGb 761 on ischemia reperfusion injury of intestine.

BACKGROUND/AIMS: The aim of this study was to determine the effect of Ginkgo Biloba (EGb 761) on reperfusion injury of the small bowel. METHODOLOGY: Forty-eight male 200-250 g Spraque-Dawley rats in six groups were used to determine the biochemical and histopathological changes after a 30-min ischemia and 30-min reperfusion. Pre-treatment with 50 mg/kg EGb 761 (Tebofortan, Karlsruhe-Germany) or 10-mL/kg saline was administered intravenously in the treatment and control groups. The superior mesenteric artery was occluded distal to the right colic artery and collateral arcades were ligated to provide complete ischemia. Ischemia was determined by the existence of pulseless or pale color of the small intestine. The return of the pulses and the reestablishment of the pink color were assumed to be the reperfusion of the intestine. Rats that were administered Egb 761 and saline were subjected to laparotomy, ischemia, or ischemia-reperfusion procedures. Mucosal lesions were graded from 0 to 5 in histopathological examination. Malondialdehyde and myeloperoxidase levels of the intestinal mucosa were measured. RESULTS: No significant difference was noted between the control and treatment groups regarding the histopathological changes. Although malonyldialdehyde and myeloperoxidase levels of the reperfusion + EGb 761 group were slightly higher than the laparotomy + saline group, they were significantly lower than the reperfusion + saline group. CONCLUSIONS: We concluded that EGb 761 pre-treatment before ischemia-reperfusion decreased malondialdehyde and myeloperoxidase levels and attenuated the mucosal damage.

Assessment of airway neutrophils by sputum colour: correlation with airways inflammation.

BACKGROUND: Airway inflammation, with recruitment of neutrophils to the airway lumen, results in purulent secretions and a variety of potential adverse consequences for patients with chronic bronchitis and bronchiectasis. We hypothesised that gradations of sputum colour would correlate directly with the myeloperoxidase content of sputum and with various other indicators of the activity and consequences of bronchial diseases. METHODS: To test this hypothesis, we quantified sputum colour by reference to a sensitive nine point colour chart and correlated this assessment with indices of a number of inflammatory mediators in sputum. RESULTS: The results indicate that standardised visual measurements of sputum colour correlated strongly with myeloperoxidase, interleukin 8, leucocyte elastase (both activity and total quantity), sputum volume, protein leak, and secretory leucocyte proteinase inhibitor (p<0.001 for all). In addition, there was a strong direct correlation between leucocyte elastase and both myeloperoxidase (p<0.003) and sputum volume (p<0.001), but a strong negative correlation with secretory leucocyte proteinase inhibitor (p<0.001). CONCLUSIONS: These results indicate that sputum colour graded visually relates to the activity of the underlying markers of bronchial inflammation. The results of this simple visual analysis of sputum provides guidance concerning underlying inflammation and its damaging potential. It also provides a useful scientific tool for improving the monitoring of chronic airways diseases and response to treatment.

Cytochemical staining and flow cytometry methods applied to the diagnosis of acute leukemia in the pediatric population: an assessment of relative usefulness.

BACKGROUND: Cytochemical staining has been used in the diagnosis of acute leukemia for more than 20 years. The general availability of flow cytometers and an extensive panel of antibody reagents useful for characterizing blood cell lineage question the usefulness of continuing routine use of the cytochemical staining for the diagnosis of acute leukemia. PATIENTS AND METHODS: Test results were evaluated in 122 (n = 122; 112 with acute lymphocytic leukemia and 10 with acute myeloid leukemia) patients selected from among 320 patients with acute leukemia at Texas Children's Hospital in 1997 and 1998. Results were selected for review if the clinical encounter represented the initial diagnostic work-up and if data were available from cytochemical staining and flow cytometry studies. RESULTS: Cell lineage classification derived from flow cytometry and cytochemical stains were in agreement in all cases. Definitive diagnoses were feasible using flow cytometry results alone in 120 of 122 patients (98.4%) as compared with only 99 of 122 patients (81.2%) when only cytochemical staining results were considered. In two patients with inconclusive flow cytometry results, cytochemical staining alone provided information sufficient for diagnosis. CONCLUSIONS: Results from this study indicate that with few exceptions, flow cytometry studies alone provide sufficient information for diagnosis and management of acute leukemia in children. Nevertheless, cytochemical staining should be available for those cases in which flow cytometry results fail to allow a definitive diagnosis. A modified testing protocol is recommended.

Pulmonary edema induced by allergen challenge in the rat: noninvasive assessment by magnetic resonance imaging.

The course of pulmonary edema formation after an intratracheal (i.t.) instillation of ovalbumin was followed noninvasively by magnetic resonance imaging (MRI) in actively sensitized Brown Norway (BN) rats. Changes in edema volume assessed by MRI mimicked the results from the analysis of the number and activation of inflammatory cells recovered from the broncho-alveolar lavage (BAL) fluid. Rats treated with budesonide did not develop edema following challenge with ovalbumin, and these animals showed a significant decrease in BAL fluid inflammatory cell numbers and eosinophil peroxidase and myeloperoxidase activities. Thus, following lung edema formation by MRI provides a reliable means of assessing pulmonary inflammation after allergen challenge. Unlike BAL fluid analysis, which requires killing animals at each time point, this method is noninvasive. MRI could be of importance for the noninvasive profiling of anti-inflammatory drugs in animal models of asthma and in the clinic. Magn Reson Med 45:88-95, 2001.

Granulocyte accumulation in ischemic/reperfused myocardium: assessment with a technetium-99m-labeled antigranulocyte monoclonal antibody in the dog.

This study tested the usefulness of technetium-99m-labeled antigranulocyte monoclonal antibody BW250/183 (AGMAb) for identifying granulocyte accumulation in ischemic/reperfused canine myocardium. In dogs with 90 minutes coronary artery occlusion and 180 minutes reperfusion (n = 8), ischemic/reperfused myocardial samples demonstrated 8.5 +/- 2.4 times more Tc-99m-AGMAb accumulation than nonischemic samples. Dogs given Tc-99m-labeled nonspecific human immunoglobulin instead of Tc-99m-AGMAb (n = 3) had about half as much accumulation (4.5 +/- 1.6, P < .05). Ex vivo myocardial imaging of Tc-99m-AGMAb demonstrated marked uptake in infarcted regions identified by absent triphenyl tetrazolium chloride staining. The amount of uptake was inversely related to the severity of ischemia (determined by radioactive microspheres) and directly correlated with tissue myeloperoxidase activity, a specific marker of granulocyte accumulation. No increase in Tc-99m-AGMAb uptake occurred in dogs with 90 minutes ischemia and no reperfusion (n = 3) or 15 minutes ischemia and 180 minutes reperfusion (n = 2). In conclusion, Tc-99m-AGMAb is taken up in reperfused infarcted myocardium by both nonspecific and specific mechanisms. Because the amount of uptake reflects myocardial granulocyte accumulation, Tc-99m-AGMAb combined with nuclear imaging techniques may be useful for studying inflammatory processes in the heart in experimental animal models and human beings.

Hematopoiesis in the liver and marrow of human fetuses at 5 to 16 weeks postconception: quantitative assessment of macrophage and neutrophil populations.

During human development, the liver and marrow both function as hematopoietic organs, but little is known about differences in the production of macrophages and neutrophils by these two organs. We used immunohistochemical stains to quantify the ratio of neutrophils to macrophages within the liver and the marrow of 16 fetuses from 5 to 16 wk postconception. At 5 wk the liver had a ratio of one granulocyte [myeloperoxidase (MPO)-positive cell] to every 9 +/- 5 (X +/- SD) macrophages (KP-1-positive cells). Between 5 and 16 wk, the granulocyte to macrophage ratio in the liver was constant, whereas it changed markedly in the marrow. Before 8 wk no MPO-positive or KP-1-positive cells were observed in bones. At 10 wk, bones still had no MPO-positive cells, but KP-1-positive cells were abundant. At 11-12 wk the granulocyte to macrophage ratio was 1 to 1 +/- 1, but by 13-16 wk it had increased to 8 +/- 3 MPO-positive cells to one KP-1-positive cell. We hypothesized that at 13-16 wk the abundance of MPO-positive cells in the marrow and their scarcity in the liver was the result of production of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSF-R) in the marrow and their absence in the liver. However, by reverse transcriptase-PCR mRNAs for G-CSF and G-CSF-R were positive in both organs at all gestations, and G-CSF and G-CSF-R proteins (by immunohistochemistry) were also abundant in all liver and marrow specimens. We then hypothesized that progenitors in the fetal liver were intrinsically different from those in the marrow, and were unable to generate clones of neutrophils. However, progenitors from the liver produced neutrophils abundantly in culture. Thus, the explanation is likely related to as yet undescribed environmental differences between the fetal liver and marrow.