Quantification of HIV-1 proviral DNA and analysis of genomic diversity by polymerase chain reaction and temperature gradient gel electrophoresis.

A competitive polymerase chain reaction/temperature gradient gel electrophoresis (PCR/TGGE) protocol was developed for exact quantification of HIV-1 proviral DNA copy numbers in clinical samples. An internal standard (ST) that differs from wildtype-sequences only by a single base exchange was used as a competitor in PCR. Quantification of HIV-1 target sequences was achieved by coamplification of defined copy numbers of ST with wild type target sequences, hybridization of PCR products to a strand-specifically labelled probe, separation of ST and wildtype sequences by TGGE, and determination of the ratio of wildtype and standard sequences by densitometric scanning. Effects of sample preparation, DNA extraction and white blood cell counts were minimized by the additional quantification of beta-globin sequences. With this technique, it was possible to determine precisely the number of HIV target sequences as compared to the number of beta-globin gene copies with a detection limit of two HIV-1 proviral copies. Forty-four peripheral blood mononuclear cell (PBMC) extracts from 39 HIV-1 infected patients were analyzed by PCR/TGGE. HIV-1 proviral DNA levels ranged between 2 and 24190 HIV-copies/10(6) beta-globin copies. In general, patients in the advanced stages of disease and/or with low CD4 counts had much higher proviral DNA levels than patients in early stages or with high CD4 counts. In patients from whom consecutive samples were obtained, progression of disease correlated with a greater than tenfold rise of HIV-copies/10(6) beta-globin copies. Compared to other recently published protocols for proviral DNA quantification, this experimental approach allows in addition direct demonstration of mutations within the amplified region. The competitive PCR/TGGE protocol described in this study is suitable for monitoring fluctuations of proviral DNA levels and to identify the genomic diversity of HIV target sequences simultaneously in one assay.

Real time in situ microscopy for animal cell-concentration monitoring during high density culture in bioreactor.

An in situ microscope (ISM) device is utilised in this study to monitor hybridoma cells concentration in a stirred bioreactor. It generates images by using pulsed illumination of the liquid broth synchronised with the camera frame generation to avoid blur from the cell's motion. An appropriate image processing isolates the sharp objects from the blurred ones that are far from the focal plane. As image processing involves several parameters, this paper focuses on the robustness of the results of the cells counting. This stage determines the applicability of the measuring device and has seldom been tackled in the presentations of ISM devices. Calibration is secondly performed for assessing the cell-concentration from the cell automated numeration provided by the ISM. Flow cytometry and hemacytometer chamber were used as reference analytical methods. These measures and the output of the image processing allow estimating a single calibration parameter: the reference volume per image equal to 1.08 x 10(-6) mL. In these conditions, the correlation coefficient between both reference and ISM data sets becomes equal to 0.99. A saturation of this system during an ultrasonic wave perfusion phase that deeply changes the culture conditions is observed and discussed. Principal component analysis (PCA) is used to undergo the robustness study and the ISM calibration step.

Nasopharyngeally and vertex recorded P300's in normal subjects.

The P300 waveform has been associated with cognitive activity during information processing, and the hippocampus has been discussed as a possible generator of this waveform. This being the case some would argue that it should be possible to record the P300 with a shorter latency and greater amplitude from an electrode placed as near the hippocampus as possible. Under local anaesthesia we inserted a specially constructed needle electrode through the left nostril in the recessus pharyngeus close to the hippocampus. The results did not support the hippocampal hypothesis. Nasopharyngeally recorded P300's were found to have significantly decreased amplitudes and extended latencies when compared to vertex recordings.

Sorption processes in gas sterilization in the medical sector.

Sorption of ethylene oxide during and after gaseous sterilization is influenced by numerous factors. It was found that ethylene oxide desorption not only depends on material to be fumigated but also to a considerable degree on the wrapping material. Although polyethylene, polyamide (nylon), polytetrafluoroethylene (Teflon), silicone, aluminum, and glass beads contained no quantities of ethylene oxide detectable by gas chromatography after 72 h of aeration, residual amounts were definitely determined, even after 76 h of aeration in polypropylene, polystyrene, polyvinylchloride, paper products, and compound products of various plastics and paper mixtures. Desorption was, in all cases, found to be better when a mixture of ethylene oxide and methyl formate was used instead of pure ethylene oxide.

Quartz exposure in the slate industry in northern Norway.

In this study we have measured exposure levels to quartz in different parts of the slate industry in Alta, Northern Norway. Full shift personal samples were collected from the breathing zones of outdoor and indoor workers in the slate quarries and a slate factory. The quartz content of respirable dust was between 7 and 41%. The slate factory had the lower quartz levels although 41% of total and 73% of respirable samples were above the Norwegian TLV for quartz. The average concentration of total quartz in the slate factory was 0.27 mg/m3 and the average concentration of respirable quartz was 0.12 mg/m3. Outdoor in the quarries the average levels of quartz were 0.58 and 0.13 mg/m3 for total and respirable quartz, respectively. From the beginning of the last decade most of the quarry-workers have built quarry halls to protect themselves against a cold winter climate. Inside in these quarry halls the average levels were 1.74 mg/m3 total quartz and 0.46 mg/m3 respirable quartz. Assessment of historical exposure showed that 32 of totally 45 quarry workers with available exposure history had a lifetime inhaled quartz dose of more than 10 g. There is reason to fear that silicosis will be an increasing problem among quarry workers if efforts to reduce quartz exposure are not put into effect.

[Blunt chest trauma with severe pulmonary contusion and traumatic myocardial infarction]. / Stumpfes Thoraxtrauma mit schwerer Lungenkontusion und traumatischem Myokardinfarkt.

In the literature the incidence of cardiac involvement in blunt chest trauma varies considerably. This reflects the diagnostic problems encountered in polytraumatised patients. We report the case of an 18 year old man who suffered bilateral pulmonary contusion and traumatic myocardial infarction following a motorbike accident. The myocardial infarct was diagnosed by means of ECG, cardiac enzymes and echocardiography. When the diagnosis was made the time for successful interventional treatment had lapsed. A coronary angiography was performed after stabilisation which revealed a proximal dilatation of the left anterior descending artery. Left ventricular function was severely impaired (ejection fraction 26%). Due to the pulmonary contusion respiratory support was required for 14 days. The course was further complicated by left ventricular failure with low output.

In vivo genetic variability of the HIV-1 vif gene.

The human immunodeficiency virus type 1 (HIV-1) vif gene encodes a 23-kDa protein (viral infectivity factor) whose exact mechanism of action is not entirely clear. Vif is believed to be highly conserved among different HIV-1 strains. We have analyzed the proviral vif sequences of 61 peripheral blood mononuclear cell samples from HIV-1 positive patients by direct solid phase sequencing and temperature gradient gel electrophoresis of polymerase chain reaction products. Inter- and intraindividual sequence variations, conserved motifs, and prevalent vif subtypes were investigated. The consensus proviral vif DNA sequence of the 61 samples as well as the consensus sequence of the 61 deduced vif amino acid sequences were found to be less conserved than previously thought. The vif proviral sequences were 58% conserved, with the 5' end of the vif gene being the most conserved region (84%). Of the vif amino acids only 45% were absolutely conserved in the 61 samples, i.e., the absolutely conserved and as such possibly functionally important domains of the vif protein comprised less than half of the vif amino acids. In two-thirds of the variable positions residues belonging to different amino acid groups were found. In individual patients the prevalent vif sequence changed with the course of disease, but the differences found in two serial samples of a patient were < or = 10%. Phylogenetic analysis revealed that one African vif subtype had been introduced in the investigated population.

Inline characterization of cell concentration and cell volume in agitated bioreactors using in situ microscopy: application to volume variation induced by osmotic stress.

A new in situ microscope (ISM) was developed and tested to perform in-line monitoring of average cell volume and cell concentration in agitated cultures subjected to osmotic stress. The ISM is directly immersed into the agitated broth in a bioreactor and generates still images of cells by using pulsed luminescent diode illumination and a virtual probe volume defined by depth of focus. This technique allows the acquisition of microscopic still images without mechanical sampling techniques. The front end of the sensor fits into a standard 25-mm port and it can be steam sterilized together with the bioreactor. The automatic image evaluation generates signals of the cell concentration and the average cell volume with a time resolution of a few minutes per data point (if a 200 MHz PC is used). Without the need for evaluation, the images can be acquired and stored at a rate of one image per 0.6 s. Hansenula anomala was cultivated as batch fermentation and monitored inline with the ISM. The ISM signal of the cell concentration agreed well with referential growth curves that were obtained from counting with a hemocytometer. The ISM signal of the average cell volume shows a gradual volume reduction as a result of the aging of the culture, and it monitors an abrupt and strong cell contraction if osmotic shocks are generated in the bioreactor. Systematic in vitro studies of osmotic shocks were performed by applying the ISM to agitated culture samples of H. anomala. The volume signal of H. anomala during osmotic shocks showed a very fast cell contraction within less than a second. Within half an hour after the shocks, no signal drifts were observed, which would indicate volume restoration. These findings suggest that the ISM volume signal can be used as an inline indicator of osmotic stress in cell cultures.

In situ microscopy for on-line characterization of cell-populations in bioreactors, including cell-concentration measurements by depth from focus.

A new technique is presented which allows the use of a front-end sensor head for in situ and on-line characterization of cell concentration and cell size during fermentation. An epifluorescence microscope is mounted in a port of a bioreactor viewing directly into the agitated broth. Still images from cells are generated using pulsed illumination. They are directly visualized on a monitor and used for automatic image analysis. The cell concentration and morphological information are determined by counting and evaluating the cell images with respect to their depth from focus characteristic. An in situ microscope was successfully tested during yeast fermentations and yielded results which correlated well with results from a hemocytometer. (c) 1995 John Wiley & Sons, Inc.

Recent infection as a risk factor for cerebrovascular ischemia.

BACKGROUND AND PURPOSE: Previous infection is discussed as a risk factor for ischemic stroke in children and younger adults. We tested the hypothesis that the role of recent infection in cerebrovascular ischemia is not restricted to younger patients and investigated which infections are mainly relevant in this respect. METHODS: We performed a case-control study with 197 patients aged 18 to 80 years with acute cerebrovascular ischemia and 197 randomly selected control subjects matched for sex, age, and area of residence. RESULTS: Infection within 1 week before ictus or examination was significantly more common among patients (38 of 197) than control subjects (10 of 197; odds ratio [OR], 4.5; 95% confidence interval [CI], 2.1 to 9.7). Patients more often had febrile and subfebrile infections (> or = 37.5 degrees C) than control subjects (29 of 197 versus 5 of 197; OR, 7.0; 95% CI, 2.5 to 20). Respiratory tract infections were most common in both groups. Bacterial infections dominated among patients but not among control subjects. Infection increased the risk for cerebrovascular ischemia in all age groups; this reached significance for patients aged 51 to 60 and 61 to 70 years. The profile of vascular risk factors was similar in patients with and patients without previous infection. Infection remained a significant risk factor when previous stroke, hypertension, diabetes mellitus, coronary heart disease, and current smoking were included as covariates in a logistic model (OR, 4.6; 95% CI, 1.9 to 11.3). CONCLUSIONS: Recent infection, primarily of bacterial origin, may be a risk factor for cerebrovascular ischemia in older as well as younger patients.