Volume 42, Issue 5

The development of atherosclerotic lesions encompasses a cascade of cellular and molecular responses that can at best be characterized as an inflammatory process, and exhibits striking similarities to autoimmune diseases, such as rheumatoid arthritis. Chemokines, cytokines and their receptors are critically involved in initiation and perpetuation of atherosclerosis, and they play important roles at all levels in the pathogenesis of this disease. In the present article, the currently available information on cytokines and chemokines as key mediators in atherosclerosis is reviewed. Furthermore, based on recent experiences of our own with very early stages of atherosclerosis, possible new ways to make use of these parameters toward improved early detection, prevention and treatment of this disease are indicated.

Activated platelets are key components in many arterial disorders. P-selectin is an activation-dependent platelet receptor, which is also identified in endothelial cells. Together with E-and L-selectin it constitutes the selectin family. These transmembrane proteins have continued to attract great interest as they support rapid and reversible cell adhesion in flow systems and thus play an essential role in multicellular interactions during thrombosis and inflammation. Similarly to other lectins, selectins bind to different glycoconjugates with varying affinities. Protein ligands, equipped with the appropriate carbohydrate and sulfate moieties for P-selectin binding, have been identified in normal peripheral blood leukocytes and several non-hematopoietic organs, as well as on cancer cells. For diagnostic purposes, P-selectin can readily be detected on the platelet surface by flow cytometry and by ELISA as a soluble ligand in the plasma. Along with other markers, these data can be used in the assessment of platelet activation status. Such results bear clinical significance since P-selectin has been implicated in the pathogenesis of widespread disorders including coronary artery disease, stroke, diabetes and malignancy.

This study analyzes the relationship between risk factors related to overweight/obesity, insulin resistance, lipid tolerance, hypertension, endothelial function and genetic polymorphisms associated with: i) appetite regulation (leptin, melanocortin-3-receptor (MCR-3), dopamine receptor 2 (D2R)); ii) adipocyte differentiation and insulin sensitivity (peroxisome proliferator-activated receptor-γ 2 (PPAR-γ 2 ), tumor necrosis factor-α (TNF-α)); iii) thermogenesis and free fatty acid (FFA) transport/catabolism (uncoupling protein-1 (UCP1), lipoprotein lipase (LPL), β 2 - and β 3 -adrenergic receptor (β 2 AR, β 3 AR), fatty acid transport protein-1 (FATP-1) and iv) lipoproteins (apoliprotein E (apoE), apo CIII). The 122 members of 40 obese Caucasian families from southern Poland participated in the study. The genotypes were analyzed by restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) or by direct sequencing. Phenotypes related to obesity (body mass index (BMI), fat/lean body mass composition, waist-to-hip ratio (WHR)), fasting lipids, glucose, leptin and insulin, as well as insulin during oral glucose tolerance test (OGTT) (4 points within 2 hours) and during oral lipid tolerance test (OLTT) (5 points within 8 hours) were assessed. The insulin sensitivity indexes: homeostasis model assessment of insulin resistance, whole body insulin sensitivity index, hepatic insulin sensitivity and early secretory response to an oral glucose load (HOMA-IR, ISI-COMP, ISI-HOMA and DELTA) were calculated. The single gene mutations such as C 105 T OB and Pro 115 Gln PPAR-γ 2 linked to morbid obesity were not detected in our group. A weak correlation between obesity and certain gene polymorphisms was observed. Being overweight (25<BMI≤30 kg/m 2 ) significantly correlated with worse FFA tolerance in male PPAR-γ 2 12Pro, LPL-H (G) allele carriers. Insulin resistance was found in female PPAR-γ 2 Pro12, TNF-α (−308A) and LPL-H (G) allele carriers. Hypertension linked to the PPAR-γ 2 Pro allele carriers was characterized by high leptin output during OLTT. We conclude that the polymorphisms we investigated were weakly correlated with obesity but significantly modified the risk factors of the metabolic syndrome.

Crohn's disease is a complex disorder, with multiple genetic traits. A frameshift mutation (Leu1007fsinsC) and two missense mutations (Gly908Arg and Arg702Trp) in the NOD2/CARD15 gene are strongly associated with susceptibility to Crohn's disease. The presence of one of these risk alleles confers a 2- to 4-fold increase in the risk of developing Crohn's disease, and the presence of two mutant alleles increases the risk over 20-fold. To facilitate the analysis of these polymorphisms, we developed three LightCycler assays to detect the missense mutations Arg702Trp and Gly908Arg and the frameshift mutation Leu100fsinsC in the NOD2 / CARD15 gene. All three assays can be run simultaneously on one LightCycler using identical cycling parameters. Analysis of 53 DNAs from Crohn's patients helped to identify carriers at allele frequencies similar to other Caucasian populations. The sequencing of such DNAs confirmed the accuracy of the assays. In conclusion, we present three rapid and robust assays to detect the Arg702Trp, the Gly908Arg and the Leu100fsinsC mutations in the NOD2 / CARD15 gene.

Cervical cancer cells were transfected with a newly discovered interleukin (IL)-18 receptor to investigate the effect of endogenous IL-18 on the regulation of immune-related factors such as Fas (CD95/Apo-1)/Fas ligand and intercellular adhesion molecules. Transfection of the IL-18 receptor selectively induced a slight enhancement of the Fas via the up-regulation of intracellular reactive oxygen species and IL-18 in cervical carcinoma C33A cells, whereas there were no effects on the expression of p53, intercellular adhesion molecules-1 and Fas ligand. Neither IL-18 receptor transfection nor recombinant IL-18 enhanced interferon-γ production in C33A cells. Thus, IL-18 receptor transfection induced IL-18 expression and enhanced intracellular reactive oxygen species and Fas expression in C33A cells in an interferon-γ-independent pathway. However, treatment with agonistic anti-Fas antibody did not induce the apoptosis of C33A/IL-18 receptor transfectants, suggesting that either reactive oxygen species play a key role in resisting the Fas-induced apoptosis of C33A cells, or Fas was not functional. These results show that C33A/IL-18 receptor cells are resistant to the apoptosis and thus can survive against the immune surveillance and activated immune cells. Our results thus suggest that IL-18 and IL-18 receptor, together, may play a role in immunoregulation or in inflammation by augmenting the levels of IL-18 and reactive oxygen species in C33A cells.

Wilson disease leads to severe hepatic and neurological pathology resulting from cellular copper overload in the respective tissue. Although the affected gene, ATP7B , has been identified, genetic testing is challenging, time-consuming and expensive. Here we describe the development and use of a novel diagnostic test for four frequent mutations (M769V, W779X, H1069Q and P1134P-fs) found in Germany and many other countries in Europe. The test is based on multiplex polymerase chain reaction and DNA strip technology and was found to be highly sensitive and specific, as well as timely and cost-effective. We conclude that this test is a useful and reliable tool to screen Wilson disease patients and their family members for these mutations and may facilitate diagnosis in this complex disease.

A considerable amount of clinical and experimental evidence now exists suggesting that many biochemical pathways strictly associated with diabetes increase the production of reactive oxygen species (ROS). However, daily moderate exercise and vitamins C and E(VCE ) supplementation can be beneficial to diabetes due to reducing blood glucose and free radical production. In the present study, we investigated the effect of VCE and moderate exercise on lipid peroxidation (MDA) and scavenging enzyme activity in the blood of STZ-induced diabetic pregnant rats. Fifty female Wistar rats were used and were randomly divided into five groups. The first and second were used as the control and pregnant control groups, respectively. The third group was the pregnant diabetic group. The fourth group was the diabetic-pregnant-exercise group. VCE-supplemented feed was given to pregnant-diabetic-exercise rats constituting the fifth group. Animals in the exercised groups were moderately exercised daily on a treadmill (16.1 m/min, 45 min/d) for three weeks (five days a week). Diabetes was induced on day zero of the study. Body weights in the five groups were recorded weekly. Plasma and red blood cell (RBC) samples were taken from all animals on day 20. Glutathione peroxidase (GSH-Px), catalase and reduced glutathione (GSH) levels in plasma and RBCs, glucose, vitamins A, E and β-carotene in plasma, the number of fetuses, and body weight were lower in pregnant diabetic rats than in control animals, whereas there was a significant increase in platelet counts and plasma and RBC MDA levels. The decreased antioxidant enzymes did not improve through exercise only. However, the decreased vitamins, and increased MDA, glucose levels and white blood cell (WBC) counts were improved either by exercise or exercise plus VCE supplementation. There were no significant changes in the RBC counts and hemoglobin values in the five groups. In conclusion, these data demonstrate that there is an increase in MDA in the blood of diabetic pregnant animals, whereas there is a decrease in the number of fetuses, antioxidant vitamins and enzymes. However, dietary VCE with moderate exercise may strengthen the antioxidant defense system due to reducing ROS. They may play a role in preventing diabetes-related diseases of pregnant subjects.

Objective: The early differential diagnosis of Alzheimer's disease (AD) remains still problematic. We developed a laboratory test enabling us to distinguish patients with AD from those with vascular dementia (VD) and healthy subjects. Methods: The AD group consisted of 22 women and 18 men. The VD group consisted of 16 women and 8 men. Age-matched controls consisted of 12 women and 9 men. Plasma pregnenolone sulfate (PregS), dehydroepiandrosterone (DHEA) and its sulfate (DHEAS) were determined by radioimmunoassay. 17-Hydroxypregnenolone (17 Preg) and 7-hydroxylated metabolites of DHEA (7αDHEA, 7βDHEA) were determined by radioimmunoassay after separation by high performance liquid chromatography (HPLC). Homocysteine (Hcy), cysteine (Cys), cysteinylglycine (Cysgly) and glutathione (GSH) were measured by HPLC. Results: The ANOVA results of significant between-group differences were as follows: The PregS and the 17-Preg and DHEAS levels were independent from the diagnosis. The 7αDHEA levels significantly depended on the sex (p<0.05) and diagnosis (p<0.01). Aminothiols were influenced by the diagnosis (p<0.01, p=0.0541, p<0.01 and p=0.0536 for Cys, Hcy, Cysgly and GSH, respectively). Using a stepwise backward regression analysis, the following parameters were obtained: X=11.5+4.03×sex+1.09× Hcy+0.190 × PregS−4.76×DHEAS+3.00×DHEA− 34.3×7αDHEA−0.885× Cysgly from which P-value as a discriminator was calculated according to the formula: P =1/(1+e −x ). Then, for P >0.5, a subject was considered as AD-positive (with 89% correct prediction). Discussion: The opportunity of early differential diagnosis of AD should help physicians to use suitable treatment for retardation of pathological processes.

HIV infection is associated with disturbances in lipid metabolism due to a host's response mechanism and the current antiretroviral therapy. The pathological appearance and progression of atherosclerosis is dependent on the presence of injurious agents in the vascular endothelium and variations in different subsets of candidate genes. Therefore, the Hha I polymorphism in the apolipoprotein E gene was evaluated in addition to triglycerides, total cholesterol, very lowd-ensity lipoprotein (VLDL), LDL, high-density lipoprotein (HDL), and apolipoprotein (apo) AI, B and E levels in 86 Brazilian HIV-infected patients and 29 healthy controls. The allele frequency for apoE in the HIV-infected group and controls was in agreement with data on the Brazilian population. Dyslipidemia was observed in the HIV group and verified by increased levels of triglycerides, VLDL and apoE, and decreased levels of HDL and apoAI. The greatest abnormalities in these biochemical variables were shown in the HIV-infected individuals whose immune function was more compromised. The effect of the genetic variation at the APOE gene on biochemical variables was more pronounced in the HIV-infected individuals who carried the apoE2/3 genotype. The highly active antiretroviral therapy (HAART)-receiving group presented increased levels of total cholesterol and apoE. Dyslipidemia was a predictable consequence of HIV infection and the protease inhibitors intensified the increase in apoE values.

Folate measurements, particularly for whole blood, show wide inter-laboratory and inter-methodology variability. This variability appears to be due in part to the lack of internationally accepted reference materials. A whole blood haemolysate, lyophilised in ampoules and designated 95/528, was therefore evaluated by 15 laboratories in five countries for its suitability as an International Standard (IS) for whole blood folate. The preparation was assayed using a variety of microbiological and protein-binding methodologies against local standards and calibrators. A consensus folate content was assigned to 95/528. The inclusion of three whole blood samples in the study with widely differing folate levels demonstrated a considerable reduction in inter-laboratory variability when the folate content of the samples was determined relative to the proposed IS 95/528 rather than to laboratories' local standards and calibrators. Accelerated degradation studies indicated that the folate content of 95/528 is stable when stored at −20°C. On the basis of the results presented here, the World Health Organization Expert Committee on Biological Standardization established 95/528 as an IS for whole blood folate.

Reliable reference ranges are important in the interpretation of laboratory data, and it is incumbent on each laboratory to verify that the ranges they use are appropriate for the patient population they serve. The objective of this study was to determine population-specific reference ranges for thyroid stimulating hormone (TSH), free thyroxine (fT4), free triiodothyronine (fT3) and total triiodothyronine (TT3) on the Abbott ARCHITECT i2000 analyzer. For this study, we used human serum samples collected from a population in Castilla y León, Spain. Serum samples were collected from 304 individuals (male, n=151; female, n=153; age 12–94 years) representing outpatients (n=100), hospitalized patients (n=104) and apparently healthy subjects (n=100). Individuals taking any medications, with a history of thyroid disorder, or severe non-thyroidal illness were excluded from the study. For healthy subjects, the following reference intervals were determined: TSH, 0.51–5.95 mIU/l; fT4, 0.84–1.42 ng/dl (10.77–18.21 pmol/l); fT3, 1.48–3.37 pg/ml (2.27–5.18 pmol/l); and TT3, 0.65–1.46 ng/ml (1.00–2.24 nmol/l). In this group, TSH and fT4 showed significant differences between men and women, but fT3 and TT3 did not. Conversely, fT3 and TT3 showed significant age-related differences, but TSH and fT4 did not. Within the outpatient group, no significant differences were seen between men and women for any of the hormones, but age-related differences were significant for fT3 and TT3. Within the hospitalized patient group, significant differences between men and women were found for TSH only, and age-related differences were significant for TSH, fT3 and TT3. Our findings are basically in accordance with previously published results for fT3, TT3 and TSH, but for fT4 our results differ from other data in the literature. This highlights the need for laboratories to confirm that the reference ranges they use are appropriate for the population they serve.

Two screening methods for the assay of amphetamines and their derivatives have been applied to the same analytical instrument for their evaluation. In addition to an assay at a cut-off of 1000 μg/l, a new specific reagent was evaluated for an ultra-sensitive assay of amphetamines and 3,4-methylendioxyme-tamphetamine with a cut-off of 300 μg/l. The assay confirmation was performed using high-performance liquid chromatography and gas chromatography/mass spectrometry techniques. The results were positive for both screening methods, confirming the efficacy of two simultaneous methods with different cut-off levels.

The aim of this study was to compare diagnostic performance of C-reactive protein (CRP) and poly-C avid ribonuclease (P-RNase) levels in the prediction of a severe clinical course of acute pancreatitis (AP). The study included 36 patients with mild and 20 with severe AP. CRP concentration was measured by an immunonephelometric method and P-RNase activity by the rate of polycytidylate hydrolysis at pH 7.8. At the time of admission, both P-RNase and CRP levels were significantly increased in all patients when compared to healthy subjects (29.2 vs. 18.7 U/l and 91.1 vs. 2.89 mg/l; p<0.001). Up to days 3 and 4 a further increase in P-RNase was observed. On the other hand, the increase in CRP continued only through days 2 and 3 (p<0.001). Severe acute pancreatitis (SAP) and mild acute pancreatitis (MAP) differed significantly with respect to P-RNase levels on all days studied; whereas CRP levels differed significantly on days 2–5 but did not differ at admission. Receiver operating characteristic (ROC) curve function analysis yielded the best sensitivity of SAP detection for P-RNase, equaling 72.2%, at the cut-off point value 65.3 U/l on day 3 after admission. The sensitivity of CRP for detection of SAP was 85.0% at 125.7 mg/l on the 2nd day after admission. Both parameters studied were significantly associated with the severity of the AP clinical course; however, on days 1 and 2 postadmission, P-RNase was more specific for detection of SAP than CRP (94.4% vs. 77.1% on the 1st day and 94.4% vs. 55.5% on the 2nd day). In conclusion, P-RNase has shown an excellent performance for early differentiation of acute necrotizing pancreatitis.

The aim of this study was to define the use of a new cardiac troponin I (cTnI) assay for emergency patients with chest pain and no specific electrocardiographic changes consistent with the presence of ischemia. Patients (n=106) admitted in Emergency/Cardiology Departments for chest pain and suspicion of acute coronary syndrome (ACS) were randomized into two diagnosis groups (ACS or non-ACS) by two independent cardiologists. cTnI measurements were performed at admission, and 6 hours and 12 hours later with a new generation assay (Access AccuTnI, Beckman Coulter). Using an upper reference limit of 0.04 μg/l, 27 patients had a cTnI elevation not related to the final diagnosis of ischemia; the positive predictive value (PPV) was 67% with specificity 48%. The decisional value was re-defined and set at 0.16 μg/l, a concentration corresponding to the 99th percentile of the non-ACS patient group. Precision (coefficient of variation) was 8% at this level, PPV 97% and specificity 98%. This new decisional value is now used in our institution and could be included in standard care guidelines to improve the management of patients presenting chest pain in emergency departments.

Novel allelic variants have been found in the glutathione S-transferase ( GST ) A1 and T1 genes. The former GSTA1*B allele is associated with low expression and the latter GSTT1*B allele lacks GSTT1 activity. The information on frequencies of both variants is poorly documented in the Japanese population. In this study we determined the frequencies of allelic variants of GSTA1 and GSTT1 in a Japanese population using PCR-restriction fragment length polymorphism and allele-specific PCR. The frequencies of GSTA1*B , GSTT1*0 and GSTT1*B alleles in the subjects were 16.0%, 71.1% and 0%, respectively. This is the first report on the frequencies of allelic variants of GSTA1 and GSTT1 in a Japanese population.

The European Communities Confederation of Clinical Chemistry and Laboratory Medicine (EC4) opened a Register for European Specialists in Clinical Chemistry and Laboratory Medicine in 1997. The operation of the Register is undertaken by a Register Committee (EC4RC). During the last 6 years more than 1500 specialists in clinical chemistry and laboratory medicine have joined the Register. In this article a Code of Conduct for Registrants which was approved at the EC4 Register Committee meeting in Amsterdam, 8 November 2003 is presented.