Long- and short-term in vitro D-dimer stability measured with INNOVANCE D-Dimer.

In vitro D-dimer stability in plasma is widely assumed, but has not yet been documented by systematic studies using samples covering a wide range of D-dimer. We investigated the short- and long-term stability of D-dimer in clinical citrated plasma samples with normal and pathological levels. The short-term stability was analysed by measuring D-dimer fresh, after storage of plasma for 4 hours at room temperature (RT) and after an additional 24 h storage at +2 to +8 degrees C (n=40). Long-term stability samples (n=40) were measured fresh and after storage for 19, 25 and 36 months at < or =-60 degrees C. The effect of repeated freezing was analysed by measuring samples (n=50) fresh and after four consecutive freeze-thaw cycles. D-dimer was measured on the BCS System using the INNOVANCE D-Dimer assay (Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany). D-dimer values at baseline ranged from 0.23-22.2 mg/l FEU. The mean percentage change after storage for 4 hours at RT and additional 24 hours at +2 to +8 degrees C was +3.8% and +2.7%, respectively. The mean percentage change after frozen storage for 19, 25 and 36 months at < or =-60 degrees C was -11.7%, -4.8% and -9.3%, respectively. The small decrease of D-dimer values after frozen storage was not time-dependent. Repeated freezing did not significantly alter D-dimer values (mean change < or =5%). The data demonstrate stability of D-dimer in plasma prior to freezing for up to 4 hours at RT and for up to 24 hours at +2 to +8 degrees C as well as in plasma stored for up to three years at < or =-60 degrees C.

Pro- and antifibrinolytic properties of human pulmonary microvascular versus artery endothelial cells: impact of endotoxin and tumor necrosis factor-alpha.

OBJECTIVE: Microvascular thrombosis is a common feature of acute inflammatory lung injury, as occurs in sepsis and acute respiratory distress syndrome, but the underlying pathomechanisms are presently not fully understood. DESIGN: Experimental. SETTING: University laboratory. SUBJECTS: Lung endothelial cells. INTERVENTIONS: We characterized the expression of tissue-type and urokinase-type plasminogen activator (t-PA and u-PA) as well as plasminogen activator inhibitor (PAI)-1 and PAI-2 in human endothelial cells (EC) from the microvascular pulmonary circulation (HMVEC-L) and compared it with that of EC from pulmonary artery (HPAEC) and umbilical vein (HUVEC) under baseline conditions and upon stimulation with either tumor necrosis factor-alpha or lipopolysaccharide. MEASUREMENTS AND MAIN RESULTS: Real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay were employed for quantification of messenger RNA and protein concentrations. Under baseline conditions, comparable PAI-1 expression was noted in all EC. HPAEC were characterized by significantly higher baseline expression of t-PA and PAI-2 compared with HUVEC and HMVEC-L. In contrast, u-PA messenger RNA concentrations were found to be significantly higher in nonstimulated HMVEC-L compared with HUVEC and HPAEC. In all EC, stimulation with tumor necrosis factor-alpha and lipopolysaccharide increased the expression of PAI-1, PAI-2, and u-PA and decreased t-PA expression. The changes in messenger RNA content were reflected by corresponding changes in the protein concentrations. CONCLUSIONS: High baseline u-PA expression is a prominent feature of human lung microvascular EC, whereas pulmonary artery EC are characterized by high t-PA concentrations. Microbial and inflammatory challenge provokes up-regulation of PAI-1 and PAI-2 and down-regulation of t-PA in both macro- and microvascular pulmonary EC, which may favor local fibrin deposition.

Statins differ in their ability to block NF-kappaB activation in human blood monocytes.

OBJECTIVE: The benefits of statin therapy in cardiovascular medicine are ascribed to its lipid-lowering effect as well as its anti-inflammatory properties. Whereas all statins have been shown to reduce cholesterol plasma levels, their effect on inflammatory markers has been inconsistent. Here, we show that statins differ markedly in their effectiveness in preventing activation of NF-kappaB, a transcription factor involved in the activation of immediately early genes during inflammation. METHODS: Six statins (atorvastatin (Atv), cerivastatin (Cer), fluvastatin (Flu), lovastatin (Lov), pravastatin (Pra), simvastatin (Sim)) were tested for their ability to influence the induction of NF-kappaB in human monocytes (Mo) during inflammation. Mo isolated from healthy blood donors were incubated with LPS (10 microg/ml) in the presence and absence of statin (0.001-5 microM). NF-kappaB binding activity (EMSA), degradation and phosphorylation of the inhibitor protein IkappaB-alpha (Western blotting), tissue factor (TF) mRNA (rtPCR), and TF activity (clotting assay) were analyzed. RESULTS: All statins inhibited LPS-induced NF-kappaB binding activity in Mo in a dose-dependent manner. The inhibitory effect was due to reduced phosphorylation and degradation of the NF-kappaB inhibitor protein IkappaB, and was primarily dependent on the absence of mevalonate. Whilst this effect appeared with all statins, there were marked differences in the degree of inhibition between the statins. Cer (45 +/- 9% inhibition, p < 0.05) was 9-fold more effective in reducing NF-kappaB activation than Flu (5 +/- 10% inhibition). The differences in the potency of statins (Cer > Atv > Sim > Pra > Lov > Flu) were also reflected at the transcriptional level and the protein level of NF-kappaB controlled tissue factor expression. CONCLUSIONS: The finding that statins differ in their potency in interfering with the activation of NF-kappaB signaling in human monocytes further supports the hypothesis that some statins inhibit the inflammatory response more than others.

Monocytes recruited into the alveolar air space of mice show a monocytic phenotype but upregulate CD14.

The evaluation of monocytes recruited into the alveolar space under both physiological and inflammatory conditions is hampered by difficulties in discriminating these cells from resident alveolar macrophages (rAMs). Using the intravenous injected fluorescent dye PKH26, which accumulated in rAMs without labeling blood leukocytes, we developed a technique that permits the identification, isolation, and functional analysis of monocytes recruited into lung alveoli of mice. Alveolar deposition of murine JE, the homologue of human monocyte chemoattractant protein (MCP)-1 (JE/MCP-1), in mice provoked an alveolar influx of monocytes that were recovered by bronchoalveolar lavage and separated from PKH26-stained rAMs by flow cytometry. Alveolar recruited monocytes showed a blood monocytic phenotype as assessed by cell surface expression of F4/80, CD11a, CD11b, CD18, CD49d, and CD62L. In contrast, CD14 was markedly upregulated on alveolar recruited monocytes together with increased tumor necrosis factor-alpha message, discriminating this monocyte population from peripheral blood monocytes and rAMs. Thus monocytes recruited into the alveolar air space of mice in response to JE/MCP-1 keep phenotypic features of blood monocytes but upregulate CD14 and are "primed" for enhanced responsiveness to endotoxin with increased cytokine expression.

Cell-based delivery of cytokines allows for the differentiation of a doxycycline inducible oligodendrocyte precursor cell line in vitro.

BACKGROUND: Stem cells, having the property of self renewal, offer the promise of lifelong repair of damaged tissue. However, somatic tissue-committed primary stem cells are rare and difficult to expand in vitro. Genetically modified stem-like cells with the ability to expand conditionally provide a valuable tool with which to study stem cell biology, especially the cellular events of proliferation and differentiation. In addition, stem cells may be appropriate candidates for therapeutic applications. METHODS: Double transgenic mice possesing SV40 T antigen (Tag) under the control of the reverse tetracycline-transactivator (rtTA) were used to establish cell lines. One brain cell line was partially characterized by DNA sequencing, morphology, antigen expression using flow cytometry, confocal microscopy, and electrophysiology using the patch clamp technique. Cell cycle analysis was performed using propidium iodide staining; cell viability and H3-thymidine incorporation assays. The ability of this cell line to differentiate was assessed by confocal microscopy following co-culture with stem cells secreting cytokines. RESULTS: We report here the establishment and partial characterization of a cell line derived from the brain tissue of rtTA-SV40 Tag transgenic mice. Analysis of the morphology and antigen markers has shown that this cell line mimics some aspects of primary glial precursors. The results of electrophysiology are consistent with this and suggest that the cell line is derived from O2A glial precursor cells. Cell cycle progression of this cell line is doxycycline-dependent. In the absence of doxycycline, cells become apoptotic. Differentiation into mature type 2 astrocytes and (precursor) oligodendrocytes can be induced upon withdrawal of doxycycline and addition of epithelial stem cells secreting cytokine, such as hIL3 (human Interleukine 3) or hIL6 to the culture. In contrast, co-culturing with hCNTF (human Ciliary NeuroTrophic Factor)-secreting epithelial stem cells did not induce them to mature into progeny cell types. CONCLUSION: The differentiation of this O2A glial precursor line does not occur automatically in culture. Additional external help is required from the cell-based delivery of appropriate transgenic cytokines. Withdrawal of doxycycline from the culture medium removes the proliferation signals and induces a fatal outcome.

Alveolar fibrin formation caused by enhanced procoagulant and depressed fibrinolytic capacities in severe pneumonia. Comparison with the acute respiratory distress syndrome.

Changes in the alveolar hemostatic balance in severe pneumonia were compared with those in the acute respiratory distress syndrome (ARDS). Analysis was performed in bronchoalveolar lavage fluids (BALF) of patients with ARDS triggered by nonpulmonary underlying events in the absence of lung infection (ARDS; n = 25), pneumonia demanding mechanical ventilation (PNEU-vent; n = 114), spontaneously breathing patients with pneumonia (PNEU-spon; n = 40), and ARDS in combination with lung infection (ARDS+PNEU; n = 43); comparison with healthy control subjects (n = 35) was performed. In all groups of patients, BALF total procoagulant activity was increased by nearly two orders of magnitude, being largely attributable to the tissue factor pathway of coagulation. Concomitantly, markedly reduced overall fibrinolytic capacity (fibrin plate assay) was noted in the lavage fluids of all patients. BALF levels of urokinase-type plasminogen activator were significantly reduced throughout, whereas the lavage concentrations of tissue-type plasminogen activator did not differ from those in control subjects. In addition, markedly enhanced levels of plasminogen activator- inhibitor I and alpha(2)-antiplasmin were noted in ARDS, ARDS+PNEU, and PNEU-vent, but not in PNEU-spon. In all groups of patients, the changes in the lavage enzymatic activities were paralleled by manifold increased BALF concentrations of fibrinopeptide A and D-dimer, reflecting in vivo coagulation processes. Within the overall number of patients with pneumonia, changes in the alveolar hemostatic balance were more prominent in alveolar and interstitial pneumonia than in bronchopneumonia. Acute inflammatory lung injury, whether triggered by nonpulmonary systemic events or primary lung infection, is thus consistently characterized by both enhanced procoagulant and depressed fibrinolytic activities in the alveolar lining layer, with the appearance of fibrin formation in this compartment. Profile and extent of changes in severe pneumonia demanding respirator therapy are virtually identical to those in ARDS, whereas somewhat less prominent alterations of the alveolar hemostatic balance are noted in spontaneously breathing patients with pneumonia.

Cationic lipids employed for antisense oligodeoxynucleotide transport may inhibit vascular cell adhesion molecule-1 expression in human endothelial cells: a word of caution.

Antisense oligodeoxynucleotides (ODN) have become a powerful tool to achieve specific gene inhibition in various cell types, including endothelial cells. The low spontaneous cellular uptake of ODN, however, usually requires the employment of transmembrane carriers, such as the positively charged liposome formulation dioleyloxypropyltrimethyl ammonium chloride/dioleoylphosphatidylethanolamine (DOTMA/DOPE). In the present study, we observed that DOTMA/DOPE per se interferes with the inducible expression of vascular cell adhesion molecule-1 (VCAM-1) in human pulmonary artery endothelial cells (HPAEC). By RT-PCR analysis, a dose-dependent suppression of VCAM-1 but not intracellular adhesion molecule-1 (ICAM-1) mRNA levels in tumor necrosis factor-alpha (TNF-alpha)-challenged HPAEC pretreated with DOTMA/DOPE (5-20 microg/ml) was demonstrated. Correspondingly, a strong reduction of TNF-alpha-induced VCAM-1 but not ICAM-1 cell surface expression on HPAEC was observed. These DOTMA/DOPE-induced changes were not due to alterations in VCAM-1 mRNA stability, nor did DOTMA/DOPE inhibit TNF-alpha-induced NF-kappaB-like binding activity in nuclear extracts of HPAEC, as analyzed by electrophoretic mobility shift assay. In contrast, DOTMA/DOPE effected a dose-dependent increase in AP-1-like binding activity in nuclear extracts of HPAEC, as analyzed by Western blotting and EMSA. We conclude that positively charged liposome preparations may per se inhibit TNF-alpha-induced endothelial VCAM-1 expression, and this may be related to changes in AP-1 but not NF-kappaB-dependent transcriptional control. Notably, when used at concentrations below 5 microg/ml, DOTMA/DOPE may be employed for specific antisense-mediated downregulation of VCAM-1 in the absence of vehicle-related side effects on adhesion molecule transcription.

A novel bacterial reversion and forward mutation assay based on green fluorescent protein.

We report the first use of green fluorescent protein (GFP) for mutation detection. We have constructed a plasmid-based bacterial system whereby mutated cells fluoresce and non-mutated cells do not fluoresce. Fluorescence is monitored using a simple hand-help UV lamp; no additional cofactors or manipulations are necessary. To develop a reversion system, we introduced a +1 DNA frameshift mutation in the coding region of GFP and the resulting protein is not fluorescent in Escherichia coli. Treatment of bacteria containing the +1 frameshift vector with ICR-191 yields fluorescent colonies, indicating that reversion to the wild-type sequence has occurred. Site-directed mutagenesis was used to insert an additional cytosine into a native CCC sequence in the coding region of GFP in plasmid pBAD-GFPuv, expanding the sequence to CCCC. A dose-related increase in fluorescent colonies was observed when the bacteria were treated with ICR-191, an agent that induces primarily frameshift mutations. The highest dose of ICR-191 tested, 16 microg/ml, produced a mutant fraction of 16 x 10(-5) and 8.8 x 10(-5) in duplicate experiments. The reversion system did not respond to MNNG, an agent that produces mainly single-base substitutions. To develop a forward system, we used GFP under the control of the arabinose PBAD promoter; in the absence of arabinose, GFP expression is repressed and no fluorescent colonies are observed. When cells were treated with MNNG or ENNG, a dose-dependent increase in fluorescent colonies was observed, indicating that mutations had occurred in the arabinose control region that de-repressed the promoter. Treating bacteria with 100 microg/ml MNNG induced mutant fractions as high as 82 x 10(-5) and 40 x 10-5 in duplicate experiments. Treating bacteria with 150 microg/ml ENNG induced a mutant fraction of 2.1 x 10(-5) in a single experiment.

Endemic institutional strongyloidiasis in British Columbia.

The incidental finding in 1984 of Strongyloides stercoralis larvae in a resident of a chronic care institution who had a vague clinical illness prompted a review of the other residents. Five other cases were identified after exhaustive laboratory investigations. Fecal-oral spread was considered the most likely manner of the spread of infection. The possibility of endemic strongyloidiasis in institutions should be considered, even in temperate climates, when there is unexplained persistent illness or high eosinophil counts. Serologic testing is a useful adjunct to fecal examination in such situations.

Discussing possible standards of natural radioactivity in building materials.

This paper discusses different possibilities of deriving reference values for the natural radioactivity concentrations in building materials to estimate possible additional radiation exposure for the population. Based on comprehensive experimental and theoretical investigations the consequences of the resulting hypothetical reference activity concentrations in building materials, applying different dose limits, were examined. The calculation of the activity concentration standards was performed for standard conditions obtained by earlier studies on exhalation of Radon-222 and Radon-220 from building materials.

Double-blind comparison of diclofensine with nomifensine in outpatients with dysphoric mood.

Depressed outpatients (n = 107, age 26-75 years) were treated with either a 50 mg single morning dose of diclofensine (n = 54) or 75-100 mg nomifensine given in two divided doses (n = 53) over a period of three weeks. The baseline mean values of the Depression Status Inventory (DSI index) of Zung corresponded to those of a mildly depressed population, as given by Zung. At the end of the treatment the mean DSI and Anxiety Status Inventory (ASI-index) values of both groups dropped to the levels of a normal population. The side-effect profile of the two treatments was similar. There were no side-effects indicating sedation. Adverse effects of the anticholinergic type were rare. It can be concluded that both diclofensine and nomifensine are beneficial for the treatment of depressed outpatients and that in a dose relation of 2:3 (diclofensine:nomifensine) they lead to a similar improvement in depressive outpatients.

Radiation exposure in German dwellings, some results and a proposed formula for dose limitation.

The results of our investigations in the Federal Republic of Germany on the indoor and outdoor exposure to natural radiation from gamma rays and radon and thoron daughters are presented. Indoor the median Rn-222 concentration was approximately four times higher than outdoors. A correlation analysis of the data obtained showed that indoors the equilibrium factor F is almost independent of ventilation, Rn-222 concentration and other parameters. The mean equilibrium factor was measured to be F = 0.3 in dwellings and approximately F = 0.4 outdoors. The results of our investigations on diffusion coefficients and exhalation rates showed, that the activity concentration in dwellings and in cellars can generally be explained by the radon exhalation from the building materials. Only in areas of high radon concentrations, the exhalation from the soil was a decisive factor. The mean effective dose equivalent by residence in dwellings amounted to 0.2 - 0.8 mSv/a for Rn-222 daughters and approximately 0.1 mSv/a for Rn-220 daughters. A relationship has been derived which permits the calculation of the expected average radiation exposure in dwellings by gamma radiation and by radon inhalation as function of the radionuclide concentration in building materials.

Proportional counter dosimetry and microdosimetry for radiotherapy with multiple pion beams.

At the Swiss Institute for Nuclear Research (SIN) cancer patients are irradiated with negatively charged pi mesons using a 60-beam medical pion generator, the Piotron. A low-pressure tissue-equivalent proportional counter was used to measure absorbed dose and microdosimetric spectra. A method was developed to allow discrimination of events from different beam components, i.e., beam contamination (electrons and muons), pions in flight, and stopping pions. Measurements were performed along the axis and at lateral distances off one of these identical pion beams. The marked changes of total microdosimetric spectra with depth in phantom detected in earlier measurements are mainly due to large variations in the dose contributions of the beam components and much less to changes in the shapes of the individual microdosimetric spectra. The single beam measurements were used to calculate three-dimensional distributions of absorbed dose and of dose mean lineal energy, yD, for dynamic patient irradiations. Within the whole target volume yD remains nearly constant when irradiated with all 60 beams, whereas considerable changes were found for irradiations with 31 beams coming from a semicircle. Both size and shape of target volumes influence yD, the maximum values ranging from 30 to 45 keV/micron.

[Expanded indications for cesarean section for low birth weight infants]. / Zur Frage der erweiterten Indikation der Schnittentbindung bei untergewichtigen Kindern.

Retrospective investigations on 849 low birth weight infants between 1,001 and 2,500 gm born from 1966 to 1982 were done. The infants were divided into three groups with birth weights from 1,001 to 1,500 gm, from 1,501 to 2,000 gm, and 2,001 to 2,500 gm. The results of spontaneous delivery, delivery with manual assistance and caesarian section were compared. In the weight group 1,001 to 1,500 gm (14%), the perinatal mortality was 32%. In caesarian sections, the perinatal mortality was 19%, in spontaneous deliveries the perinatal mortality was 32% and in delivery with manual assistance the perinatal mortality was 45%. In the second weight group of 1,501 to 2,000 gm the perinatal mortality was 14.8%. Spontaneous delivery had a perinatal mortality of 16%, manual assistance a perinatal mortality of 14% and caesarian section a perinatal mortality of 10%. In both birth weight groups of 1,001 to 1,500 gm and 1,001 to 2,000 gm, the infants born by caesarian section had a significantly improved prognosis. Therefore, we recommend caesarian section in these weight groups for all breech presentations. In vertex presentations, caesarian sections are indicated when additional risk factors especially prolonged labour are apparent. In the third weight group between 2,001 and 2,500 gm the overall perinatal mortality was 4.9%. The spontaneously delivered infants have a mortality of 4.1%. Manual assistance was associated with a perinatal mortality of 5.9%. Caesarian section had a perinatal mortality of 11%.(ABSTRACT TRUNCATED AT 250 WORDS)

Tiaprofenic acid overdose.

After ingestion of 2000 mg tiaprofenic acid (Surgam), a 14-year-old girl was admitted to the clinic. The ingested dose was tolerated without signs of systemic intoxication. Tiaprofenic acid, its metabolites, and products of chemical decomposition were identified by gas chromatography/mass spectrometry in the urine sample of the patient. Additionally, a previously unknown decomposition product of a metabolite was detected. For quantitation of the drug in plasma, a rapid and sensitive high-performance liquid chromatography assay was established.

Age dependent concentration of 226Ra in human bone and some transfer factors from diet to human tissues.

The concentration of 226Ra in more than 1000 human bone samples, still born infants, fetuses, blood, mixed diet, drinking water and mineral water has been measured. An age dependence of 226Ra concentration in human bone could be demonstrated. Up to an age of 20 yr two maxima of 226Ra concentration in bone occur, coinciding with the increased velocity of skeleton growth. Normally, the main source for uptake of radium by man is food. In Germany the transfer factor from diet to bone is about 0.098. Compared with data from literature for other countries, mainly the U.S.A., the German value is higher by a factor of about 1.5. This difference may be due to the lower calcium uptake in Germany which leads to higher resorption of radium. Two methods for measuring natural radium concentrations is biological material and water samples are briefly described: a gamma-gamma-coincidence method for indirect measurement of 226Ra via the short lived 214Bi(RaC) and an alpha-spectrometric method for direct measurement of the alpha-emission of 226Ra.

Recent results of the German Thorotrast study--epidemiological results and dose effect relationships in Thorotrast patients.

The German Thorotrast Study includes 5159 Thorotrast patients and 5160 control patients. 887 Thorotrast patients and 660 control patients could be clinically and biophysically examined and followed-up. The mean age at injection or hospitalization in the case of the control group was 28 yr. The mean injected volume of Thorotrast was calculated to be 24.7 ml and the X-ray films of 249 Thorotrast patients showed paravascular deposits. In the meantime 432 Thorotrast patients and 122 patients of the control group have died. Among the deceased patients we have registered (Thorotrast vs control): hepatic tumors 152/0; myeloproliferative diseases 10/0; Hodgkin's diseases 2/0; non-Hodgkin's lymphomas 5/1; bronchogenic carcinomas 13/6; pleural mesothelioma 1/0; bone sarcoma 1/1(?); sarcoma at injection site 1/0; hepatic cirrhosis 90/6; bone marrow failure 8/1; other neoplastic diseases 46/19; other non-neoplastic diseases 151/88. The cumulative incidence of liver tumors depends on the dose rate to liver tissue and is not influenced by the age at injection. A dose effect relationship for the myeloproliferative diseases is not yet apparent.