Fluorescence flow cytometry of human leukocytes in the detection of LDL receptor defects in the differential diagnosis of hypercholesterolemia

A flow-cytometric method with fluorescence-labeled monoclonal antibodies (MABs) against the low density lipoprotein (LDL) receptor (C7A MAB) or 3,3'-dioctadecylindocarbocyanin-iodide (DiI) LDL has been developed that allows the quantification of LDL receptors on leukocytes and the identification of patients with familial hypercholesterolemia (FH) within 48 hours. Leukocytes were isolated from 10 mL anticoagulated blood by density gradient centrifugation. To induce maximal expression of LDL receptors, mononuclear cells were preincubated with either phytohemagglutinine (PHA) or lipoprotein-deficient serum (LPDS). LPDS-treated monocytes provided a more homogeneous cell population with regard to LDL receptor activity than did the PHA-treated lymphocytes; they also provided a greater discrimination between the fluorescence of the receptor probes and cellular autofluorescence. The C7A MAB was able to compete for DiI LDL binding by about 40%. In competition with unlabeled LDL, DiI LDL revealed linear binding, indicating an affinity similar to native LDL. The binding characteristics of DiI LDL were also similar to 125I-LDL binding. LDL isolated from familial defective apolipoprotein B-100 was not able to compete for DiI LDL binding on monocytes, whereas native LDL reduced it by about 80%. In monocytes from FH heterozygous patients, the cellular mean fluorescence using either C7A MAB or DiI LDL at 4 degrees C was 30% to 70%; in FH homozygotes, cellular mean fluorescence was less than 20% of that in monocytes from normal individuals. In patients with familial defective apolipoprotein B-100 antibody binding was normal, but one patient's own LDL failed to compete with normal DiI LDL for 4 degrees C binding on U937 test monocytes. Patient monocytes having internalization defects showed normal 4 degrees C DiI LDL binding, but at 20 degrees C cell-associated fluorescence was reduced by about 40%. In our study 384 hypercholesterolemic patients (preselected according to serum cholesterol levels, clinical symptoms, and family history) were analyzed for LDL receptor expression using the C7A MAB-based assay. In 71.8% of the patients with cholesterol levels higher than 300 mg/dL, an LDL receptor deficiency was observed. Apolipoprotein E isoforms and lipoprotein[a] were found to be independent from the LDL receptor status. In some patients with high cholesterol levels but normal LDL receptor expression with the C7A MAB assay, LDL receptor defects could be diagnosed when either reduced binding or internalization of DiI LDL or familial defective apolipoprotein B-100 was detected.(ABSTRACT TRUNCATED AT 400 WORDS)     

MRT of experimental liver abscesses: a comparison of a new blood pool contrast medium with gadolinium-DTPA in animal experiments

PURPOSE: In an experimental pyogenic liver abscess model, the signal intensities were compared intraindividually and interindividually after the application of a new blood pool contrast agent, 24-gadolinium-DTPA (diethylenetriamine-pentaacetic acid) cascade polymer, and after the application of gadopentetate dimeglumine. METHODS: In 20 rabbits with experimentally induced liver abscesses, the relative signal intensities of the liver, abscess centre, abscess wall and portal vein were assessed before and between 30 seconds and 60 minutes after injection of a 25 mumol/kg dose of gadolinium polymer and of 100 mumol/kg of gadolinium-DTPA, respectively. Measurements were performed at 1.5 Tesla, using a head coil and a Flash-2-D sequence. RESULTS: The interindividual comparison (unpaired T-test, p < 0.05) yielded significant differences of the relative signal intensities of the abscess centre (at any time point after contrast-media application), abscess wall (between 15 and 60 minutes after contrast media application), and portal vein (between 30 seconds and 7.5 minutes after contrast media application). The interindividual comparison showed a significantly higher abscess centre-liver contrast (between 30 seconds and 12.5 minutes after contrast media application) and a significantly higher abscess wall-centre contrast (between two and 7.5 minutes after contrast media application) after the application of gadolinium polymer compared with gadopentetate dimeglumine. CONCLUSION: In this animal model, the higher abscess centre-liver contrast after the application of gadolinium polymer was the basis for a better and prolonged visibility of the abscesses, as compared with images acquired after injection of gadopentetate dimeglumine.     

Survival of Yersinia enterocolitica and Escherichia coli in spring water

Survival of pure and mixed cultures of Yersinia enterocolitica and Escherichia coli in sterile spring water stored at 4 degrees C was evaluated. Y. enterocolitica increased two or three logs during the first 3 weeks of incubation, and at the end of 64 weeks viable cells in numbers corresponding to the initial inoculum level were still detectable. However, after 1 week of incubation, the number of E. coli in water started to decrease and in 13 weeks time no E. coli was detected. More rapid decrease of E. coli was observed in mixed cultures.     

Prebiotic synthesis of sequential peptides on the Hadean beach by a molecular engine working with nitrogen oxides as energy sources

Addressing the still open question of the prebiotic origin of sequential macromolecules (peptides, nucleic acids) on the primitive Earth, we describe a molecular engine (the primary pump), which works at ambient temperature and continuously generates, elongates and complexifies sequential peptides. This new scenario is based on a cyclic reaction sequence, whose keystep is the activation of amino acids into their N‐carboxyanhydrides (NCA) through nitrosation by NOx. This process could have taken place on tidal beaches; it requires a buffered ocean, emerged land and a nitrosating atmosphere. With the help of geochemical studies and computer simulations of atmosphere photochemistry, we show that the primitive Earth during the Hadean may have satisfied all these requirements. © 2001 Society of Chemical Industry.     

Lack of de-epoxidation of type B trichothecenes in incubates with human faeces

The ability of human gastrointestinal organisms to transform the trichothecenes 3-acetyldeoxynivalenol and nivalenol was investigated. Samples of human faeces were incubated under anaerobic conditions for 48 h with the toxins. They were then extracted and analysed for trichothecenes and metabolites. 3-Acetyldeoxynivalenol was metabolized to deoxynivalenol during the incubation period. In contrast to what has been reported for other species such as rats, mice and pigs, no de-epoxidated metabolites were detected in the faecal incubates. The toxicological significance of the difference in the intestinal ability to transform trichothecenes between species is unknown.     

Clinical investigation of traumatic injuries of permanent incisors in Izmir, Turkey

470 injured teeth of 370 patients who consulted the Dental Clinic of Ege University, Izmir, Turkey for examination or treatment between 1981-1993 were evaluated. Information concerning sex, age of patients at the time of injury, cause of trauma, number of injured teeth, type of tooth and type of trauma were recorded. More boys suffered traumatic injuries (64.8%) than girls (35.2%). Patients aged 11-15 years old exhibited the highest number of injuries (34.4%) followed by the 6-10 years old group (24.5%). Most injuries involved one tooth (60%) and maxillary central incisors were the most often affected teeth (66.2%). The leading cause of injury was undefined falls (45.1%). At the initial examination, cases seen after a long posttraumatic period showed more complications than those presented within a short time period. Educational programs about the importance of dental trauma, the benefits of immediate attendance and conservation of avulsed and fractured teeth would be very helpful for patients. Additionally improving the knowledge of the dental practitioner about trauma would be another important point in solving the problem.     

Secure vehicular communication systems: implementation, performance, and research challenges

Vehicular communication systems are on the verge of practical deployment. Nonetheless, their security and privacy protection is one of the problems that have been addressed only recently. In order to show the feasibility of secure VC, certain implementations are required. In [1] we discuss the design of a VC security system that has emerged as a result of the European SeVe- Com project. In this second article we discuss various issues related to the implementation and deployment aspects of secure VC systems. Moreover, we provide an outlook on open security research issues that will arise as VC systems develop from today?s simple prototypes to fullfledged systems.