Volume 42, Issue 2

Matrix metalloproteinases (MMPs), also called matrixins, are proteinases that participate in extracellular matrix remodelling and degradation. Under normal physiological conditions, the activities of MMPs are precisely regulated at the level of transcription, of activation of the pro-MMP precursor zymogens and of inhibition by endogenous inhibitors (tissue inhibitors of metalloproteinases; TIMPs). Alteration in the regulation of MMP activity is implicated in diseases such as cancer, fibrosis, arthritis and atherosclerosis. The pathological effects of MMPs and TIMPs in cardiovascular diseases involve vascular remodelling, atherosclerotic plaque instability and left ventricular remodelling after myocardial infarction. Since excessive tissue remodelling and increased matrix metalloproteinase activity have been demonstrated during atherosclerotic lesion progression (including plaque disruption), MMPs represent a potential target for therapeutic intervention aimed at modification of vascular pathology by restoring the physiological balance between MMPs and TIMPs. This review describes the members of the MMP and TIMP families and discusses the structure, function and regulation of MMP activity; finally, pharmacological approaches to MMP inhibition are highlighted.

Activated protein C (APC), a natural anticoagulant, is formed from protein C by the action of the thrombinthrombomodulin (TM) complex on the endothelial cell surface. Endothelial protein C receptor augments the activation of protein C by the thrombin/TM system. APC inactivates the activated form of coagulation factors V and VIII in the presence of protein S. Administration of APC reduced the pulmonary vascular injury and hypotension as well as the coagulation abnormalities by inhibiting production of the tumor necrosis factor-α (TNF-α) in rats given endotoxin (ET). These therapeutic effects of APC could not be attributed to its anticoagulant effects. APC inhibited ET-induced TNF-α production in human monocytes by inhibiting activation of nuclear factor k-B and activator protein-1 in vitro. Administration of the human plasma-derived APC ameliorated coagulation abnormalities without any adverse effects in patients with disseminated intravascular coagulation (DIC). Recombinant APC was reported to reduce the mortality of patients with severe sepsis, and the therapeutic effect was more marked in such patients with overt DIC than those without it. These observations strongly suggest that APC plays important roles in the regulation of inflammation as well as coagulation. Both anti-inflammatory and anticoagulant properties of APC might contribute to the therapeutic usefulness in patients with severe sepsis.

HER2/ERBB2 protein is a 185 kDa transmembrane growth factor receptor whose extracellular domain, a 105 kDa fragment (p105), can be released from cell surfaces by proteolytic cleavage. The aim of our study was to compare serum p105 concentrations in normal and pathological pregnancies and to determine whether any correlation exists between preeclampsia and p105 levels. Serum p105 was assayed in 96 nonpregnant women and 89 pregnant women (26 normotensive, 14 normotensive with a history of preeclampsia or fetal hypotrophy, 10 with chronic hypertension, 10 with gestational hypertension and 29 with preeclampsia). Median serum p105 levels (median; 95% confidence interval) were higher in the preeclampsia group (13.9 μg/l; 12.8–16.1 μg/l) than in the normotensive (11.7 μg/l; 10.6μ13.3 μg/l; p<0.05) or non-pregnant groups (9.3 μg/l; 8.9–9.6 μg/l; p<0.001). There were no significant differences between the other pregnancy groups. In the normotensive group, serum p105 was correlated with the number of gestations (r=0.46; p<0.05), parity (r=0.39; p<0.05) and placenta weight (r=0.61; p<0.05). In preeclamptic women, serum p105 correlated with parity (r=0.46; p<0.05). Serum p105 concentrations above 11.9 μg/l were associated with a high odds ratio (OR) for onset of preeclampsia (after adjustment for parity OR=9.0; 95% CI=2.3–36.0; p<0.005). Preeclampsia is associated with increased serum p105 concentrations, which may be related to increased fetomaternal cell traffic.

Carbonyl stress compounds such as glyoxal and methylglyoxal have been recently attracting much attention because of their possible clinical significance in chronic and age-related diseases. A high-performance liquid chromatographic procedure has been developed for the simultaneous quantitation of glyoxal and methylglyoxal in human urine. The assay is based on the reaction of these compounds with 1,2-diamino-4,5-dimethoxybenzene to form fluorescent adducts, which are separated by reversed-phase highperformance liquid chromatography in a total run time of 45 minutes and quantitated fluorometrically using 2,3-pentanedione as an internal standard. Derivatization is performed for diluted urine (100–120 mOsm/kg H 2 O) under acidic conditions (pH 4.5) at 60°C over a prolonged time (15 h) to maximize the yields. The assay is specific and sensitive enough to analyze urinary levels of glyoxal and methylglyoxal with the within-and between-day relative standard deviations of less than 5%. Urinary levels (mean±standard deviation, n=16) of glyoxal and methylglyoxal in healthy subjects were 4.7±1.35 μg/mg creatinine, 2.2±0.65 μg/mg creatinine, respectively, the former being 2 to 3 times more than the latter in every subject. The glyoxal and methylglyoxal levels positively correlated with each other, which may suggest that the levels reflect the individual activity of glyoxalase by which both compounds are detoxified.

Hyperthyroidism is a hypermetabolic state accompanied by increased oxygen utilization, increased production of reactive oxygen species and consequentially measurable changes in antioxidative factors. Therefore, the activities of whole blood glutathione peroxidase (GPx) and erythrocyte superoxide dismutase (SOD), total antioxidant status (TAS) in serum and erythrocytes, and serum urate and transferrrin concentrations were determined in 70 women: 14 with newly diagnosed Graves' disease (group A); 28 with hyperthyroidism on therapy with methimazole (group B, divided into two subgroups, B1 and B2) and 28 healthy women (group C). In comparison with control group C, GPx activity was significantly decreased in all patient groups (p<0.05), whereas SOD activity was significantly decreased in group A (p<0.01) and significantly increased in group B (p<0.01). In comparison with the control group, serum TAS activity was significantly decreased in group A, and erythrocyte TAS activity in all patient groups. Study results suggest that the impaired antioxidative factor balance leads to the development and presence of oxidative stress in women with hyperthyroidism. The severity of these alterations, considered contradictory by some authors, appears to depend on the use of therapy.

The aim of the present investigation was to evaluate the diagnostic accuracy of brain natriuretic peptide (BNP) and amino terminal proBNP (NT-proBNP) for the detection of mild/moderate and severe impairment of left ventricular ejection fraction (LVEF). In 180 subjects BNP and NT-proBNP were measured by two novel fully automated chemiluminescent assays (Bayer and Roche methods). LVEF as determined by echocardiography was categorized as normal (>60%), mildly/moderately reduced (35–60%) and severely diminished (<35%). Discriminating between patients with LVEF<35% (n=32) and subjects with LVEF ≥35% (n=148), receiver-operating characteristic (ROC) curve analysis revealed an area under the curve (AUC) of 0.912 for BNP and of 0.896 for NT-proBNP (difference 0.016, p=0.554). In contrast, BNP displayed an AUC of 0.843and NT-proBNP an AUC of 0.927 (difference of 0.084, p=0.034) when comparing patients with LVEF 35–60% (n=37) and individuals with LVEF >60% (n=111). Evaluation of discordant false classifications at cut-off levels with the highest diagnostic accuracy showed advantages for BNP in the biochemical diagnosis of LVEF<35% (4 misclassifications by BNP and 25 by NT-proBNP, p<0.001) and for NT-proBNP in the detection of LVEF 35–60% (25 misclassifications by BNP and 7 by NT-proBNP, p=0.002). In conclusion, the present study indicates a different diagnostic accuracy of BNP and NT-pro-BNP for the detection of mildly/moderately reduced LVEF and severely diminished LVEF. Advantages of BNP may be advocated for the biochemical diagnosis of more severely impaired LVEF, while NT-proBNP might be a more discerning marker of early systolic left ventricular dysfunction.

Oxidized low-density lipoproteins (ox-LDLs) and their autoantibodies (OLAB) are involved in the development of atherosclerosis in animal models, but their role in humans is still not clear. For this reason we studied 54 patients with β-thalassemia major (TM), as a model of chronically low circulating LDLs with a high level of oxidation; 44 patients with primary hypercholesterolemia, as model of chronically high circulating LDLs; 24 type 2 diabetic mellitus patients (T2DM) before and after 3 months of atorvastatin treatment (20 mg/day), as a model of acute changes in circulating LDLs; and 41 normolipidemic subjects as a control group. ox-LDLs were measured by the determination of baseline diene concentration in the plasma LDL lipidic fraction after 12 hours fasting and were expressed as the amount of conjugated dienes/liter (BDC/l) or BDC/LDL-cholesterol (LDL-C), which indicate respectively LDL oxidation degree and status. OLABwere determined using an enzyme immunoassay and related to LDL oxidation degree (BDC/l). In TM, BDC/l was lower, while BDC/LDL-C was significantly higher, compared to both hypercholesterolemia and normolipidemic subjects. Patients with hypercholesterolemia had higher BDC/l, but lower BDC/LDL-C and OLAB/BDC-l, than normolipidemic subjects. In T2DM patients at diet, BDC/LDL-C and OLAB/BDC-l were lower than in normolipidemic subjects. After 3 months of atorvastatin treatment, BDC/LDL-C and OLAB/BDC-l ratios increased. When all patients were evaluated together, a significant inverse correlation was evident between OLABand either LDL or BDC/l. Our findings suggest that a relationship between OLABtiter and oxidation indices (BDC/l and BDC/LDL-C) does exist and we may speculate that an increase in OLAB/BDC-l ratio might be protective against the risk of atherosclerosis.

β 2 -Glycoprotein I (β 2 GPI) is the major target molecule for so-called anticardiolipin antibodies. We evaluated the isolation procedure of β 2 GPI from human plasma with special emphasis on the time of precipitation, composition of different isolated fractions and their antigenic properties. The isolation was initiated by perchloric acid precipitation for either 3, 18 or 50 min, followed by heparin affinity and cationic exchange chromatography. The properties of isolated proteins were tested by rocket electrophoresis, enzyme-linked immunosorbent assay, polyacrylamide gel electrophoresis, immunoblotting and N-terminal sequencing. Each isolation procedure, regardless of the perchloric acid precipitation duration, resulted in three distinct protein peaks, differing in protein composition qualitatively. Comparing sequential peaks between the isolations of different precipitation times, we found that all the three first peaks (set of peaks No. 1), all the three second peaks (set No. 2) as well as the three third peaks (set No. 3) consisted of identical proteins but in different quantities. Set No. 1 was composed of immunoglobulins and a lesser amount of β 2 GPI. In set No. 2 only unnicked β 2 GPI was detected. Protein C inhibitor was found in addition to smaller amounts of unnicked β 2 GPI in set No. 3. Oxidation or degradation of β 2 GPI during the isolation procedure did not result in a mixture of different forms of β 2 GPI but rather in a lower yield of wild-type β 2 GPI. The co-existence of β 2 GPI and protein C inhibitor in the isolated fractions may suggest their protein-protein interactions in vivo.

Insulin-like growth factor-I (IGF-I) is known as an important stimulator of collagen and glycosaminoglycans (GAGs) biosynthesis in tissues. IGF-I activity is under control of IGF-I-binding proteins (IGFBPs) with different IGF-I-binding affinity. IGFBP-1 is known as an inhibitor of IGF-dependent functions. Some IGFBPs (e.g., IGFBP-1) may undergo phosphorylation that dramatically increases IGFBP affinity for IGF. Wharton's jelly represents a reservoir of IGF-I and its binding proteins (BPs). Pre-eclampsia, the most common, pregnancy-associated pathological syndrome, contributes to a significant decrease in IGF-I and IGFBP-1 content in Wharton's jelly, although it does not affect collagen content in this tissue. In the present study we show that control Wharton's jelly contains phosphorylated forms of IGFBP-1 that are dramatically dephosphorylated during pre-eclampsia. A dramatic decrease in IGF-I binding to immunoprecipitated IGFBP-1 from pre-eclamptic Wharton's jelly compared to the control was observed. Western immunoblot analysis with anti-phosphothreonine antibodies for immunoprecipitated IGFBP-1 from control and pre-eclamptic Wharton's jelly revealed that both tissues contain phosphorylated forms of IGFBP-1. However, a distinct decrease in the expression of phosphorylated IGFBP-1 from pre-eclamptic Wharton's jelly was observed. The functional significance of the phenomenon was found in cultured fibroblasts treated with IGFBP isolated from Wharton's jelly extracts. A significant decrease in collagen biosynthesis was found in the cells treated with IGFBP of control Wharton's jelly, while in the presence of IGFBP from pre-eclamptic Wharton's jelly, the rate of collagen biosynthesis was similar to that in the control cells. The result was corroborated by data showing increase in expression of IGF-I receptor and phosphorylated MAP kinases (ERK1 and ERK2) in fibroblasts cultured in the presence of IGFBP from pre-eclamptic Wharton's jelly, compared to control. The data suggest that the decrease in phosphorylated IGFBP-1 in pre-eclamptic Wharton's jelly may decrease IGF-I-binding affinity for IGF and increase the bioavailability of IGF-I for receptor interaction. This mechanism may facilitate IGF-I-dependent stimulation of fibroblasts to produce extracellular matrix (ECM) components even at a low IGF-I tissue level. Therefore, IGFBP-1 phosphoisoforms in Wharton's jelly may play an important role in the regulation of IGF-I-dependent functions during pre-eclampsia.

Background and purpose: Several studies show a link between microalbuminuria and markers of inflammation. Increased urine albumin excretion accompanies acute ischaemic stroke. The mechanisms responsible for microalbuminuria in acute stroke patients remain unclear. The goal of this study was to determine if there is a relationship between urine albumin excretion and interleukin-6, a key mediator of acute phase reaction. Methods: Nineteen acute ischaemic stroke patients and 13 controls matched for age, sex and cardiovascular risk factors were included. Serum interleukin-6 level was measured on the 2nd day of stroke at 06:00 am, 10:00 am, 06:00 pm and 10:00 pm using ELISA method. Urine albumin excretion was determined using immunonephelometric method. Results: Interleukin-6 level and urine albumin excretion were significantly higher in stroke patients than in controls. There was a significant correlation between interleukin-6 level and urine albumin excretion in stroke patients (R=−0.60 at 06:00 am; R=−0.49 at 10:00 am; R=−0.64 at 06:00 pm; R=−0.54 at 10:00 pm) but not in controls. Conclusions: Urine albumin excretion in acute ischaemic stroke is related to serum interleukin-6.

Background: 24S-Hydroxycholesterol (24OHC) and 27-hydroxycholesterol (27OHC) are two structurally similar oxysterols of different origins–the former almost exclusively formed in the brain and the latter formed to a lesser extent in the brain than in most other organs. Hypothesis to be tested: Neuronal damage and/or demyelination causes increased flux of 24OHC from the brain into the cerebrospinal fluid (CSF), whereas a defect blood-brain barrier causes increased flux of 27OHC from the circulation into the CSF. Methods: Isotope dilution-mass spectrometry was used to assay the two oxysterols in CSF and plasma from more than 250 patients with different neurological and geriatric diseases. Results: The CSF-levels of the two oxysterols were much more affected by the different diseases than the plasma levels. Patients with active demyelinating diseases had increased levels of 24OHC in CSF with a relatively high 24OHC/27OHC ratio. Patients with meningitis in general had high levels of both steroids with a low 24OHC/27OHC ratio. Patients with Alzheimer's disease had slightly increased levels of 24OHC in CSF with less increase in 27OHC. Patients with multiple sclerosis had a tendency to have higher levels of 24OHC during active periods with a high 24OHC/27OHC ratio. Conclusions: Measurements of the two oxysterols in CSF and plasma may add significantly to existing biochemical methods for evaluation of neurological diseases.

This study was designed to test the hypothesis that impaired neutrophil function might contribute to the development of bacterial pneumonia in patients with HIV-infection. Numbers of inflammatory cells and immunoglobulin G Fcγ receptor (IgG FcγR) I, II, III levels were investigated in bronchoalveolar lavage (BAL) fluid of HIV-seronegative and HIV-infected patients with bacterial pneumonia. The 99 patients were classified into three groups: I: HIV-seronegative and pneumonia (n=40); II: HIV-infected and pneumonia (n=19); III: HIV-seronegative with other pulmonary diseases than pneumonia (n=40). The results of groups I and II, II and III, and I and III were compared. The percentage of alveolar macrophages was significantly lower (group II vs. III: p=0.005, group I vs. III: p=0.001), that of neutrophils increased significantly in patients with pneumonia (group II vs. III: p=0.02, group I vs. III: p=0.01). Lymphocytes differed only between groups I and III (p=0.04). Although only the expression of FcγRI was significantly higher in HIV-seronegative pneumonia patients compared to those without pneumonia (p=0.01), the mean expression of all three receptors was lower in the HIV-infected group, with that of FcγRI approaching statistical significance. This report provides first evidence that altered FcγR expression on BAL neutrophils might contribute to the increased susceptibility of HIV-infected patients to bacterial pneumonia.

A novel statistical approach has recently been described to estimate diagnostic “errors” in method comparison studies. This procedure requires a population-based probability, p p , which describes the spread of values within the population under study, and an analytical probability, p a , quantifying the risk of errors at a decision limit due to replacing one method by the other. The population probability was derived from 207 subjects who were submitted to an oral glucose tolerance test due to suspicion of type 2 diabetes. The new concept was then applied in a reverse mode by determining the analytical variability of glucose concentrations for a fixed discordance rate diagnosing type 2 diabetes. If a combined discordance rate (sum of positive and negative discordances) of 5% is allowed, a maximal imprecision of 3.7% can be tolerated in the absence of bias. In the presence of a 3.0% bias, the allowable imprecision must be reduced to 2.8%. The relationship between bias and imprecision followed a complex function and not a simple linear model. These allowable limits were achieved with venous plasma in the fasting state. The allowable analytical specifications were slightly more stringent with capillary blood. After a 2 h glucose challenge, higher error rates could be tolerated, indicating that post-load glucose concentrations have a higher diagnostic efficiency than fasting levels. The new concept has the advantage that it is derived from patient's samples in relation to diagnostic requirements.

The association of hyperhomocysteinemia and hypercysteinemia with the risk of arterial and venous thrombosis is well documented. While it is known that standardized pre-analytical conditions are necessary for reliable measurement of plasma total homocysteine, the effects of pre-analytical conditions on cysteine measurement are less well known. The aim of this study was to evaluate the effects of pre-analytical conditions on the measurement of homocysteine and cysteine. We observed that the concentration of total homocysteine in plasma increased significantly with time (38% after 6 h), whereas total cysteine decreased (5% after 2h) when blood anticoagulated with ethylenediaminetetraacetic tripotassium salt was kept at room temperature. These changes were minimized when acidic citrate dextrose was used as an anticoagulant and were abolished when blood samples were immediately placed on crushed ice, independently of the anticoagulant. Storage of plasma for 72h at room temperature induced a small (≅6%), but significant, decrease in cysteine when blood was collected in ethylenediaminetetraacetic tripotassium salt. In contrast, homocysteine was stable in plasma for 72h, independently of the anticoagulant used. In conclusion, if blood samples for plasma total homocysteine and cysteine measurement cannot be kept on ice, they should be collected in acidic citrate dextrose to minimize the artifactual changes.

We analyzed 95 sera, demonstrating interference in a previous study, with the Kryptor homogeneous time-resolved fluorescence resonance energy transfer carcinoembryonic antigen (CEA) immunoassay (Brahms AG, Berlin, Germany). Only one serum differed, i.e., 6.0 μg/l for Kryptor vs. 13.3 μg/l for a microtiter plate in-house immunofluorometric assay (IFMA), using both aggregated mouse immunoglobulins as blocker and capture monoclonal antibody (Mab) F(ab′) 2 -fragments to reduce interference. Sera were further analyzed with experimental IFMAs with intact capture Mabs and without blocker. The Kryptor-CEA assay Mab GFR44 (capture) had elevated results in 71% of sera with the Kryptor Mab G15 (tracer) or in 81% with our tracer Mab. G15 (capture) had 65% elevated results with GFR44 (tracer) or 99% with our tracer Mab. The latter was reduced from 99% to 62% by adding Kryptor blocker to the buffer or to 30% by adding our blocker. Finally, combining both assay Mabs and assay buffer from Kryptor still gave interference in 16% of sera. Kryptor-CEA assay results thus agreed with our inhouse CEA assay results, showing no interference. As the Kryptor-CEA assay antibodies were sensitive to interference and the Kryptor-CEA assay buffer did not reduce interference as efficiently as our in-house assay buffer, the Kryptor-CEA assay format was crucial for the absence of interference.

Linearity evaluation of an analytical method is important for both manufacturers of diagnostic devices and laboratory users. Some of the statistical assumptions for estimation and testing in linear regression are violated in analytical methods that count particles per unit of volume or/and time, leading to potential erroneous evaluation of linearity. The objective of this paper is to provide an approach for evaluating linearity in these cases. The number of counts for each concentration level has a Poisson probability distribution that is linear, second-, or higher-order polynomial function of the concentration. Maximum likelihood approach is used to estimate the parameters of the models. Deviance of a particular model and the likelihood ratio test are used to test for linearity. An evaluation of linearity of an analytical method in multiple experiments is also described. No particular changes to the standard testing protocols and data collections are necessary. There are several statistical software packages that can perform the calculations. Formulas and SAS codes presented in this article can also assist in estimation and statistical testing.

The carcinogenic potential of environmental xenoestrogens (i.e., organochlorines) is a matter of controversy. Their pathogenic role as promoters in breast cancer has been previously suggested. In Europe, despite their prohibition since the ’70s, residues persist in soil and rivers resulting in a widespread contamination of the general population. In this study, we have compared the serum levels of p,p'-1,1-dichloro-2, 2-bis (4-chlorophenyl) ethylene (DDE) and hexachlorobenzene (HCB) in 231 women at the time of breast cancer discovery and in 290 age-matched healthy controls. p,p'-DDE was found in 76.2% of cases and in 71.1% of controls but HCB was present only in 12.6% of cases (29 from 231) and in 8.9% of controls (26 from 290). Even if taking all undetectable results (recorded as “0”) into consideration, mean values were significantly different in cases when compared to controls. The serum level of p,p'-DDE was 3.46±3.48 ppb (0.58±0.58 μg/g lipid) in patients and 1.85±2.09 ppb (0.31±0.35 μg/g lipid) in controls (p<0.0001). The HCB serum level was 0.66±1.25 ppb (0.11±0.21 μg/g lipid) in patients and 0.20±1.02 ppb (0.03±0.17 μg/g lipid) in controls (p<0.0001). When considering p,p'-DDE and HCB as binary variables (1 if higher than the limit of quantification, 0 if lower), the presence of both residues was significantly associated with an increased risk of breast cancer development (OR 2.21, 95% CI 1.41–3.48 for p,p'-DDE and OR 4.99, 95% CI 2.95–8.43 for HCB). No excess was observed among parous women or when familial history of breast cancer was considered. In the cancer group, no differences in serum levels of p,p'-DDE or HCB were found in relation with estrogen-receptor (ER) status, Bloom stage or lymph node metastasis, but the HCB level was moderately correlated with tumor size (p=0.026).

A fluorescence polarization immunoassay (FPIA) has been commercially released for routine large-scale testing of total homocysteine (tHcy) on the AxSYM analyzer. We evaluated the analytical performance of the AxSYM tHcy FPIA and compared it with the well established high-performance liquid chromatography (HPLC) and IMx tHcy FPIA methods. Homocysteine concentrations were measured by AxSYM and IMx tHcy FPIA and by a rapid isocratic HPLC method with fluorescence detection. Coefficient of variation (CV) of total imprecision for AxSYM tHcy was ≤5%, mean dilution recovery 102%, analytical sensitivity 0.70 μmol/l and linearity was good up to 1:8 dilution. Spearman rank correlations, rho, were 0.83 (p<0.0001) for AxSYM vs. HPLC, 0.97 (p<0.0001) for AxSYM vs. IMx and 0.83 (p <0.0001) for IMx vs. HPLC. Passing and Bablok regression Y-intercepts and slopes were: 2.944/0.937 (AxSYM vs. HPLC), −0.367/1.142 (AxSYM vs. IMx) and 2.632/0.805 (IMx vs. HPLC). Corresponding mean differences (AxSYM-Comparison Assay) recorded over a 5–50 μmol/l measured range were 1.80, −0.73 and 2.53 μmol/l. AxSYM tHcy FPIA's first rate precision, supported by the complete automation of the AxSYM analyzer, makes it fit for routine use and suitable for laboratories requiring homocysteine high-throughput testing capabilities.