Activated transcription factor nuclear factor-kappa B is present in the atherosclerotic lesion.

Nuclear factor-kappa B (NF-kappaB)/Rel transcription factors play an important role in the inducible regulation of a variety of genes involved in the inflammatory and proliferative responses of cells. The present study was designed to elucidate the implication of NF-kappaB/Rel in the pathogenesis of atherosclerosis. Activation of the dimeric NF-kappaB complex is regulated at a posttranslational level and requires the release of the inhibitor protein IkappaB. The newly developed mAb alpha-p65mAb recognizes the IkappaB binding region on the p65 (RelA) DNA binding subunit and therefore selectively reacts with p65 in activated NF-kappaB. Using immunofluorescence and immunohistochemical techniques, activated NF-kappaB was detected in the fibrotic-thickened intima/media and atheromatous areas of the atherosclerotic lesion. Activation of NF-kappaB was identified in smooth muscle cells, macrophages, and endothelial cells. Little or no activated NF-kappaB was detected in vessels lacking atherosclerosis. Electrophoretic mobility shift assays and colocalization of activated NF-kappaB with NF-kappaB target gene expression suggest functional implications for this transcription factor in the atherosclerotic lesion. This study demonstrates the presence of activated NF-kappaB in human atherosclerotic tissue for the first time. Atherosclerosis, characterized by features of chronic inflammation and proliferative processes, may be a paradigm for the involvement of NF-kappaB/Rel in chronic inflammatory disease.

Increased IkappaB kinase activity is associated with activated NF-kappaB in acute myeloid blasts.

NF-kappaB/Rel transcription factors are modulators of immune and inflammatory processes and are also involved in malignancy. Phosphorylation of the IkappaB inhibitors by the IkappaB kinase (IKK) complex leads to their proteasomal degradation, resulting in activated NF-kappaB. Here, we investigated the activation status of NF-kappaB and the IKK complex in acute myeloid leukemia (AML). Gelshift assays revealed an increased level of activated nuclear NF-kappaB in myeloid blasts. Both bone marrow and peripheral blood blasts from AML patients showed enhanced IKK activity relative to controls, whereas the IKK protein concentrations were comparable. In addition, an increased level of IkappaB-alpha was detected in AML blast cells, although this appeared to be insufficient to block nuclear translocation of NF-kappaB, also confirmed by immunofluorescence. In subtype M4 and M5 AML cells a more extensive NF-kappaB activation and higher IKK activity was found than in M1/M2 specimens. Isolated AML blasts cultured ex vivo responded to external stimulation (TNF, LPS) by further IKK activation, IkappaB degradation and NF-kappaB activation. Preincubation with the proteasome inhibitor PSI inhibited the NF-kappaB system in isolated AML blasts. This study established for the first time a dysregulation of IKK signaling in AML leading to increased NF-kappaB activity suggesting potential therapeutic avenues.

Effect of proteasome inhibitors on monocytic IkappaB-alpha and -beta depletion, NF-kappaB activation, and cytokine production.

We investigated the effect of proteasome inhibitors on the lipopolysaccharide (LPS)-induced expression of several monocytic cytokines, which may be dependent on the transcription factor, nuclear factor-kappaB (NF-kappaB). Exposure of human monocytic THP-1 cells to ALLN and Mu873 prevented the LPS-induced degradation of IkappaB-alpha and -beta, as did the more potent proteasome inhibitor, PSI, whereas several calpain inhibitors were ineffective. This was accompanied by the inhibition of nuclear NF-kappaB binding activity and NF-kappaB transcriptional activation. At the mRNA level, the inhibitors blocked the expression of tumor necrosis factor (TNF) and interleukin-1beta (IL-1beta), whereas IL-8 remained unaffected by ALLN and was only partially reduced by the highest dose of PSI. The latter effect appears to be due to an increase in IL-8 mRNA stability in the presence of proteasome inhibitors. Furthermore, the production of TNF was efficiently suppressed by ALLN and PSI, less by Mu873, and not at all by calpain inhibitors. In primary human blood monocytes ALLN also prevented the LPS-induced degradation of IkappaB-alpha and -beta, efficiently blocked the production of TNF and, to a lesser extent, IL-1beta, whereas that of IL-8 was not inhibited. The expression of NF-kappaB-dependent monocytic cytokines may be selectively controlled by the proteasome, offering a potential therapeutic target in inflammatory disease.

Expression of messenger RNA of the cardiac isoforms of troponin T and I in myopathic skeletal muscle.

In the absence of clinical signs, elevated values of the cardiac isoforms of troponin T (cTnT) and I (cTnI) can be found in the serum samples of some patients with skeletal muscle myopathies; the cause is unclear. We studied the messenger RNA (mRNA) expression of cTnT and cTnI in the skeletal muscles of 24 patients with histologically proven myopathies and in 18 patients in whom a myopathy could be excluded. For cTnT- and cTnI-mRNA determination, we designed specific primer pairs for nested polymerase chain reaction. After amplification, the products were digested with 2 restriction enzymes and visualized. We found cTnT mRNA in 7 skeletal muscle biopsy specimens (6 patients with Duchenne muscular dystrophy, 1 patient with a primary sarcoglycanopathy) and cTnI mRNA in 6 (5 with Duchenne muscular dystrophy, 1 patient with a histologically negative biopsy). The mRNA of the cardiac isoforms, cTnT and cTnI, is expressed in the skeletal muscles of patients with Duchenne muscular dystrophy, but also in some other myopathies. Further studies are needed to show whether the mRNA is translated into the protein, but serum levels of cTnT and cTnI in patients with Duchenne muscular dystrophy would seem to indicate this.

A data processing system adapted to the special needs of the emergency laboratory.

A data processing system for the emergency laboratory was integrated in our clinical laboratory computer system, its prime objective being the service requirements of the laboratory. It included the possibility of simultaneous optical reading of request forms and on-line capturing, processing, and printing of laboratory test data. Priority request forms, which allow the clinician to specify the interval by which emergency test results must be available, are registered by an optical reader and arranged according to urgency by the computer. The production of worksheets is replaced by visual display of information required for accurate specimen analyses on a large colour TV screen. The individual processing status of all tests from as many as 30 request forms is displayed in a colour code. For process control the updated delay time for test performance is faded in. All reports are produced by direct machine transfer of verified test results. For security purposes all steps of sample processing (request, result, report) are recorded via line printers outside the emergency laboratory. The capacity of the computer for managing sample and data processing reduces the work load for technicians. This results in a reduction of the turn-round time of tests. 95% of all requested tests are performed and reported within the requested time period and in emergencies, test results are available within 5-10 min. There has been no major breakdown of the system in over one year of use.

Radioimmunoassay for subunit B in isoenzymes CK-MB and CK-BB of creatine phosphokinase.

We report the development of a double-antibody system for radioimmunoassay of CK-B subunit in isoenzymes CK-MB and CK-BB of creatine phosphokinase. With our method, 4.1 ng/ml of isoenzyme CK-BB can be detected. Within-run and between-run coefficients of variation are respectively 5.3% and 19.6% Analytical recovery of added antigen is 98.9 +/- 14.7%. The CK-B concentration in sera from 20 healthy adults was less than 75 ng/ml. In sera from 25 patients with a rise in CK-total activity, but without CK-MB or CK-BB activity tested with the previous reported immunological methods, we found CK-B concentrations in the range 0--86 ng/ml. In contrast, in 23 sera from patients with acute myocardial infarction within the last 48 hours CK-B concentration was in the range 104--225 ng/ml.

Clinical value of cystatin C determination.

It has been reported that cystatin C (cys-C) is elevated in patients with malignant disease. In order to investigate whether this phenomenon is linked to or independent of renal function, and at the same time examine the role of this marker in other pathological situations, cys-C concentrations were compared with 24-h creatinine clearance values in three groups of patients; the first group were undergoing treatment for malignant disease, the second group were renal transplant patients and the third randomly taken from patients for whom a routine creatinine clearance had been requested. Several patients with malignant disease had high cys-C levels without any correspondence to creatinine clearance values. Additionally, although cys-C shows a high sensitivity for detecting impaired glomerular function in renal transplant patients, the specificity was very low, with little discrimination being observed between patients with normal and pathological creatinine clearance levels. In other patients both the sensitivity and specificity of cys-C could be shown to be very good. Thus although cys-C can generally be recommended as a marker of the glomerular filtration rate, there are some patients for whom the clinical relevance is unclear.

Determination of creatine kinase isoenzyme MB activity in serum using immunological inhibition of creatine kinase M subunit activity. Activity kinetics and diagnostic significance in myocardial infarction.

This is a new method for the determination of creatine kinase isoenzyme MB activity in serum. The method uses direct activity measurement of creatine kinase B subunit activity after blocking of CK-M subunit activity by inhibiting antibodies. The test takes no longer than 15 min. The method yields an intra-serial C.V. of 2.0-12.9%, and a C.V. from day to day of 5.5%. The detection limit is 3.4 U/l creatine kinase MB. In the 95 cases with proven myocardial infarction several types of creatine kinase MB activity kinetics could be determined. The percentage of creatine kinase MB of peak CK-total is 6-25%, with a mean of 11.1%. The amount of creatine kinase MB with respect to total CK activity after reinfarction is higher than the amount after initial infarction.

Simultaneous occurrence of the MB isoenzyme of creatine kinase and macro creatine kinase type 1 in a patient with acute myocardial infarction.

When the immunoinhibition method is used for differentiation of isoenzymes of creatine kinase, the simultaneous occurrence of the MB isoenzyme and variants of macro creatine kinase may lead to misinterpretation in the diagnosis of acute myocardial infarction. We describe the case of an elderly woman in whom the MB isoenzyme and the type 1 variant of macro creatine kinase were found simultaneously in her serum after acute myocardial infarction. It proved possible to identify the MB isoenzyme rapidly using a fast enzymeimmunoassay.

Results of the multicentre evaluation of an electrochemiluminescence immunoassay for HCG on Elecsys 2010.

This study evaluated the performance of the HCG STAT Elecsys assay in 8 European laboratories using the Elecsys 2010 system. Analytical sensitivity was < 0.5 mlU/mL. The analysis of concentration series prepared by mixing serum pools with high and low HCG concentrations proved linearity up to 10.000 mIU/mL. A high-dose hook effect was not seen up to HCG concentrations of 430.000 mIU/mL. The medians of the within-run CVs (n = 21, 3 series) were 3.0% (2.1-5.8% CV; 10.4-14.4 mIU/mL), 2.4% (1.7-6.1% CV; 35.6-88.6 mIU/mL) and 2.3% (1.7-6.1% CV; 282.3-643.8 mIU/mL). The medians of the between-day imprecisions (n = 10-21) were 7.0% CV (5.2-12.0% CV; 4.0-14.0 mIU/mL), 5.5% CV (3.1-7.2% CV; 35.4-92.7% mIU/mL) and 4.1% CV (2.8-5.1% CV; 270.8-658.0 mIU/mL). The median recovery of two external quality control samples with assigned values of 9.39 and 10.40 mIU/mL) were 101.2 and 104.3% (ranges: 94.8-116.1%, 98.6-117.8%, n = 10). The assay was compared with five non-isotopic automated routine immunoassay systems (x). Slopes ranged from 0.87 to 1.15 and intercepts from-0.53 to 12.50 mIU/mL. The coefficients of correlation were with one exception (0.898) > or = 0.960. The distribution of HCG in samples from non-pregnant women and healthy men was very similar to that observed with other automated routine methods. The HCG Elecsys assay is very specific for the intact holo-hormone. Nicked HCG dimer, nicked and non-nicked beta-subunits are weakly recognised or not detected. Hemoglobin, bilirubin and lipemia (tested up to: Hb, 3.7 g/L; bilirubin, 500 mumol/L; triglyceride, 37.6 mmol/L) did not interfere the assay. The HCG Elecsys assay is well suited for the early and fast diagnosis of normal pregnancy and the detection of tubal pregnancy.

Elecsys TSH, FT4, T4, T-uptake, FT3 and T3. Clinical results of a multicentre study.

6 assays for the assessment of thyroid function (TSH, FT4, T4, T-uptake, FT3 and T3) were targets of the International Multicenter Study on the random access analyzer Elecsys 2010. The aim of the study was to characterize the clinical performance of the assay in method comparison and reference range studies. The assays under evaluation were compared to a broad variety of radio isotopic and non-radio isotopic assays. They are suitable for serum and plasma samples. In case of TSH the study include 2nd and 3rd generation TSH procedures. In general, good to excellent correlations were found between the Elecsys and the respective routine methods. Systematic deviations were extraordinary low in case of TSH, FT4 and T4. Regarding the analysis of T3 and FT3 some systematic deviations in terms of standardization have been observed. Results of Elecsys T4 and Elecsys FT4 were independent of the serum total protein or serum albumin concentrations. In T3 and FT3 Elecsys the results of samples from NTI (non-thyroidal-illness) patients were decreased, reflecting the physiological situation in these patients. Studies using samples from healthy euthyroid as well as untreated hypo- and hyperthyroid individuals enabled us to assess the assays reference ranges.

Elecsys CEA, PSA and AFP. Clinical results of a multicentre evaluation.

Three tumormarker assays, Elecsys CEA, PSA and AFP, have been evaluated in an international multicentre study to characterize their clinical performance and to verify the comparability with the corresponding tests of the Enzymun-Test product line and other methods. For each of the markers results were obtained from four laboratories. On the basis of 314 and 199 specimens respectively, (preliminary) reference ranges could be established for CEA and PSA. For the prostate marker, the age dependence of the antigen level could be clearly confirmed. Mean concentrations range between 0.51 ng/ml (< 40 years) and 3.57 ng/ml (> 70 years). Referring to CEA, 95th percentiles of 4.31 ng/ml and 2.69 ng/ml were elaborated for smokers and nonsmokers. In general, good to excellent correlations (r > 0.98) were found between the Elecsys and Enzymun-Tests. Regarding the systematic comparability of both systems, most of the slopes derived from the individual method comparison studies are within the +/- 10% range of the respective standardization results. The specific distribution pattern of the individual tumormarker values elaborated with sample material of known clinical background, reflects the well established categorization of different benign and malignant diseases according to their characteristic marker levels. Of utmost importance, however, is the excellent comparability of the Elecsys assays with the corresponding Enzymun-Tests and the FDA approved AIA 1200 tests from TOSOH in follow-up studies. Almost superimposable concentration curves guarantee that identical diagnostic information is derived from all three methods. Especially for PSA, a series of measurements on sera of prostatectomized patients proved the usability and clinical value of the test also for this particular indication. For either one of the Elecsys tests, the feasibility of using plasma as sample material was verified.

Evaluation of a new chemiluminescence immunoassay for the determination of human chorionic gonadotropin in serum.

A new commercially available chemiluminescence immunoassay for the quantitative measurement of human chorionic gonadotropin and its beta-subunit in serum was compared with the enzyme immunoassay used in our routine laboratory. Human chorionic gonadotropin was determined in serum from pregnant women, as well as from women with abortus imminens, suspected ectopic pregnancies or with molar pregnancies. The new human chorionic gonadotropin assay was also evaluated in combination with an automatic sample processor for distributing samples to the antibody-coated wells of the microtitre plates. The analytical precision, specificity and accuracy of the human chorionic gonadotropin assay were assessed with 152 sera, using the 60 min-incubation as well as the shorter 15 min version. Specificity was comparable with the conventional system, whereas the chemiluminescence assay performed better with respect to the assay detection limit and measuring range. The enhanced chemiluminescence system for the determination of human chorionic gonadotropin is an efficient assay which agrees well with our routine assay. In connection with an automatic sample processor it enables an advanced and versatile system for the determination of human chorionic gonadotropin in laboratories with large series. The system is rapid, easy to handle and apparently free from interference.

[Optimization of sample splitting in a centralized clinical chemical institute (author's transl)]. / Optimierung der Probenverteilung in einem zentralisierten klinischen-chemischen Institut.

Mechanized sample splitting machines controlled by a laboratory data processing system have been realized in only a few centralized laboratories. Bottlenecks and mistakes in sample processing are avoided by means of direct machine readable identification and parallel splitting of the secondary tubes. Our previous experience has shown that a strategy for sample splitting has to go far beyond these basic functional requirements. The splitting process must be suited to the organization of the particular laboratory, and it must be adjusted to deal with problems of individual samples containing analytically interfering substances, or variable splitting may be required in cases of inadequate sample volumes. In addition to sample identification, the secondary tube has to be coded with the date and the type of material. This allows cumulative on-line processing and series of analyses of different materials. A suitable positional arrangement of containers for control material must be born in mind for quality control performances. We have realized these additional requirements by means of a consequent mutual adaptation in the layout of the request form (marking of priority and additional information), the file structure of the data processing system and the control program of the sample splitting machine.

A rapid assay for the quantification of myoglobin: evaluation and diagnostic relevance in the diagnosis of acute myocardial infarction.

We evaluated a new, fast, quantitative, turbidimetric assay (TurbiTimeSystem, Behringwerke AG, Marburg, Germany) for the determination of myoglobin concentration in serum. Within-run imprecision (n = 10) was < 3.7% in controls ranging from 81.1 to 621.4 micrograms/l and between-day imprecision (n = 50) was < 6% in controls ranging from 69.5 to 623.4 micrograms/l. The assay is linear over the measuring range and interfering substances such as bilirubin, haemoglobin or haptoglobin do not interfere but triacylglycerol-rich samples are only measurable after brief ultracentrifugation. EDTA- or citrate-treated samples display depressed myoglobin concentration when compared with serum samples. The upper reference limit for apparently healthy individuals (n = 100, 50 female and 50 male) is 61.5 micrograms/l. Comparison with nephelometry revealed a good correlation (r = 0.982) between the two methods with the regression equation: turbidimetric assay = 5.53 + 1.02x nephelometric assay. Serial determination of myoglobin concentration and creatine kinase in 18 patients with proven acute myocardial infarction showed in general an equal diagnostic significance for both analytes. In the first 4 hours after onset of chest pain, the determination of myoglobin can have an advantage, since it is released into the blood stream at an earlier stage, but thereafter myoglobin can lead to false negative diagnosis. Therefore, determination of creatine kinase and its isoenzyme MB is still the diagnostic strategy of choice in the diagnosis of acute myocardial infarction.

Isocratic ion-pair high-performance liquid chromatographic methods for the determination of uroporphyrin and coproporphyrin type II and IV isomers in human urine.

Urinary porphyrins of porphyric patients were isolated as their methyl esters by using a simple, modified thin-layer chromatographic system. Existing methods for the isocratic ion-pair high-performance liquid chromatographic separation of uroporphyrin and coproporphyrin isomers were decisively improved by elevating the column temperatures, changing the types of columns used and modifying the eluent compositions. These techniques were applied to the determination of the isomeric distribution of uroporphyrins and coproporphyrins isolated from urines of patients in the acute or latent phase of acute intermittent porphyria. In these urines relatively high contents of the atypical uroporphyrins II (2-5%) and IV (13-19%) were found. The coproporphyrin fractions contained significantly smaller amounts of the atypical isomers II (1-2%) and IV (2-5%), the presence of which was demonstrated for the first time in such urines. Several mechanisms for the formation of the atypical coproporphyrin isomers are discussed. The isocratic ion-pair separation method served also to control the isomeric purity of uroporphyrin specimens of both natural and synthetic origin.