A simple and rapid approach to prepare Sindbis and West Nile viral RNA controls for differentiation between positive samples and laboratory contamination.

Reverse transcription-polymerase chain reaction (RT-PCR) is frequently used for surveillance and diagnosis of arboviruses and emerging viruses. A disadvantage of RT-PCR assays, especially nested assays, is the potential for false-positive results caused by laboratory contamination from either positive controls or positive samples. Positive reactors usually require sequence determination for confirmation which delay timeous reporting of a result. Thus, the aim of the study was to use a simple technique to prepare a positive control allowing true positives to be differentiated from laboratory contamination based on size differentiation for conventional PCR, or melt temperatures for real time assays. A flavivirus positive control and an alphavirus positive control were prepared for two RT-PCR assays that we are currently using for arbovirus surveillance in South Africa. Primers targeting a region of the partial genes of interest cloned in pGEM®T-easy were modified at the 5' ends with non-viral nucleotides. The resulting amplicons were circularised, resulting in pGEM®T-easy constructs with 51 and 65 non-viral bases inserted into the partial flaviviral and alphaviral genes respectively and used as template for transcribing RNA. Sequence analysis was used to confirm the manipulation of the partial genes. Using virus specific primer pairs, viral RNA could be readily differentiated from the modified positive controls either by size differentiation, or melt temperature in a SYBR®Green real time RT-PCR. This study demonstrates how simple recombinant technology can be used to produce a positive control that has application in the laboratory for surveillance studies or as a diagnostic tool using synthetic genes to abrogate the requirement for handling infectious virus.

Metaproteome analysis reveals that syntrophy, competition, and phage-host interaction shape microbial communities in biogas plants.

BACKGROUND: In biogas plants, complex microbial communities produce methane and carbon dioxide by anaerobic digestion of biomass. For the characterization of the microbial functional networks, samples of 11 reactors were analyzed using a high-resolution metaproteomics pipeline. RESULTS: Examined methanogenesis archaeal communities were either mixotrophic or strictly hydrogenotrophic in syntrophy with bacterial acetate oxidizers. Mapping of identified metaproteins with process steps described by the Anaerobic Digestion Model 1 confirmed its main assumptions and also proposed some extensions such as syntrophic acetate oxidation or fermentation of alcohols. Results indicate that the microbial communities were shaped by syntrophy as well as competition and phage-host interactions causing cell lysis. For the families Bacillaceae, Enterobacteriaceae, and Clostridiaceae, the number of phages exceeded up to 20-fold the number of host cells. CONCLUSION: Phage-induced cell lysis might slow down the conversion of substrates to biogas, though, it could support the growth of auxotrophic microbes by cycling of nutrients.

Investigation of charge ratio variation in mRNA - DEAE-dextran polyplex delivery systems.

mRNA pharmaceuticals represent a new class of therapeutics, with applications, in cancer vaccination, tumour therapy and protein substitution. Formulations are required to deliver messenger RNA (mRNA) to the target sites where induction of genetic transfection following receptor mediated cell uptake & translation is required. In the current study, the cationic polysaccharide diethylaminoethylen (DEAE) - Dextran was selected as a model system carrier for the investigation of polyplex nanoparticle formation together with mRNA as a function of the molar ratio of the components. The structure of the mRNA/Dextran colloids was investigated as a function of the polymer-to-mRNA ratio and correlated with the biological activity determined by cellular transfection with luciferase coding mRNA. Dynamic light scattering (DLS), small angle x-ray scattering (SAXS), and small angle neutron scattering (SANS) with deuterium contrast variation were used to achieve structural insight into the systems. Similarly to previously investigated lipid based systems, colloidally stable particles with confined size were obtained with either excess of positive or negative charge. Highest activity was obtained with positive charge excess. From the scattering experiments information on the internal organization inside the polymer/mRNA systems was derived. Indication for the presence of structural elements in the length scale of ten to 20 nm were found in the excess of dextran, which could be due to either excess or particulate polymer. Information on the molecular organization of the mRNA nanoparticle products may provide a valuable basis for defining critical quality attributes of drug products for pharmaceutical application.

Infrared thermography of the pig thorax: an assessment of selected regions of interest by computed tomographical and anatomical parameters.

Current methods of diagnosis of respiratory diseases in swine are invasive, time-consuming and expensive. Infrared thermography (IRT) of the thorax might provide a new method of high specificity to select swine affected with lung alterations for further diagnostics. In this study, layer thickness of different tissues was determined in frozen thorax slices (FTS) by computed tomography (CT) and then related to skin temperatures measured by IRT in healthy pigs. The aim was to determine appropriate regions of interest (ROI) for evaluation of IRT images. Organ layer thicknesses measured in CT images correspond to those measured in FTS. Temperature differences between lung ROIs and abdomen ROIs were positively correlated with lung layer thickness at certain localizations, and negatively correlated with the thickness of the thorax wall and of inner organ layers. Reference values of differences between skin temperatures were established for two ROIs on the thorax with potential practical use for lung health status determination. Respective ROIs were located on vertical lines crossing the 7th (right) and the 10th (left) thoracic vertebrae. The presence of ribs affected skin temperature significantly.

[Facial perforation trauma: an unusual injury in a skiing accident]. / Perforatationstrauma im Gesichtsschädel : Eine ungewöhnliche Verletzung beim Skifahren.

Facial perforation injuries are very rare. We describe a case of a 48-year-old man who sustained a perforation trauma from an 11 cm long wooden tree branch in the middle of the face in a skiing accident. He suffered from additional injuries, such as fractures of the ribs and hand, but was neurologically without pathologic findings and was cardiopulmonary stable.The branch penetrated the head from the sinus maxillaris through the maxilla just missing the internal and external carotid arteries and ending just short of the cervical vertebra. The patient was transported to a center for oral and maxillofacial surgery and underwent several operations.He could return to his normal social and professional life 8 months after the accident.

[Rare injury from blunt abdominal trauma in a child]. / Seltene Verletzung bei stumpfem Bauchtrauma im Kindesalter.

Presented in this work is a rare injury of a blunt abdominal trauma in a child. Besides a partial rupture of the kidney and a retro-/intraperitoneal haematoma, a further injury occurred from the accident: an initially clinically indetectable tear of the A. iliaca communis which was found intraoperatively and with systematic CT analysis. Traumatic blood vessel lesions of the abdominal aorta and in particular the iliac blood vessels are very rare in children. By such violent impact injuries, it is therefore vital to perform a clinical examination of the foot pulse, systematic analysis of radiology diagnostics, and intraoperative exploration. The growth phase should be considered for therapy of the blood vessels depending on the child's age group. As the long-term results of graft implants are practically unknown, if possible a primary suture or vein patch should be performed.

Magnetic Resonance Female Breast Imaging (MRFBI) - Evaluation of the Changes in Signal Intensity over Time Pre- and Post-administration of 0.2 mmol/kg Gd-DTPA.

PURPOSE: We are looking for a threshold value to discriminate between benign and malign breast lesions in MRI of female breast after administration of 0.2 mmol Gadolinum-DTPA/kg bw. MATERIALS AND METHODS: Double coil breast MRI (1.5 Tesla) were performed in 65 patients with an suspicious lesion for malignancy in an anteriorly examination. 57 patients could be evaluated in our study design. Histopathological 35 patients had an invasive carcinoma, 3 patients had an in-situ-carcinoma and in 27 patients benign changes were found. RESULTS: For different carcinoma diameters we found a different increase of signal intensity (SI): small carcinoma (< 10 mm) had an maximum increase of SI of 102 %, medium sized (10 to 20 mm) 222 % and carcinomas over 20 mm showed an increase of 271 %. We did not find a significant difference between SI in benign and malign lesions. The sensitivity was 94.6 % the specificity 65 %. CONCLUSION: A threshold value to distinguish between malign and benign in MRI could not be defined. With the double normal Gd-DTPA dose we do not have better specificity and sensitivity than for normal dose (0.1 mmol/kg bw) is described.

[Cutting speeds and success of biopsy with different punch biopsy instruments]. / Schnittgeschwindigkeiten und Biopsieerfolg unterschiedlicher Stanzbiopsieinstrumente.

PURPOSE: The goal of our investigations was the verification of the relationship between the reachable cutting speed and the biopsy success for different large-core needle biopsy devices in dependence of the cutting depth as well as different kinds of tissue. METHODS: We measured the dynamic cutting properties of five large-core needle devices with moveable outer needles in air and different tissues. An incremental stripe had been attached on to the outer needle and was moved through a photooptical sensor during the cutting phase. For the valuation of the biopsy success we compared the absolute weight respectively the volume of the tissue probes as well as the relative weight in relation to the size of the sample chambers within the inner trocars. RESULTS: A clear correlation between the cutting speed, the tissue properties and the biopsy success had been registered, especially for more solid tissues. CONCLUSIONS: For a successful large-core needle biopsy a cutting speed of 8 to 12 metres per second is essential. To compensate slower needle movements high quality requirements for the surface, geometry and edge have to be fulfilled.

Rapid enrichment and detection of melanoma cells from peripheral blood mononuclear cells by a new assay combining immunomagnetic cell sorting and immunocytochemical staining.

Commonly used methods for detection of melanoma cells in blood, including RT-PCR and immunocytochemistry, display only a limited sensitivity and specificity. Reliable detection of less than one melanoma cell per ml of blood is hardly possible using these methods. To obtain greater sensitivity so that a single melanoma cell in up to 25 ml of blood can be detected (5 x 10(7) peripheral blood mononuclear cells, or PBMC), we developed a new assay for combined enrichment and immunocytochemical detection of disseminated melanoma cells from PBMC of patients with malignant melanomas. Melanoma cells are directly magnetically labeled using colloidal superparamagnetic microparticles approximately 60 nm in diameter conjugated to the anti-melanoma monoclonal antibody 9.2.27, with no reactivity to normal cells in blood. Magnetically labeled melanoma cells are enriched from PBMC by magnetic cell separation and detected by a new approach for immunocytochemical staining with monoclonal mouse anti-melanoma antibodies (anti-MelanA and HMB-45). The efficiency of this assay was demonstrated in a model system in which 5-500 tumor cells from the melanoma cell line SK-MEL-28 were seeded into PBMC samples from healthy donors containing 5 x 10(7) leukocytes. Mean recovery of the seeded tumor cells was 47.4 +/- 13.99% (n = 15). Applying the assay to 20-50 ml blood samples of patients with stage III-IV malignant melanomas, we were able to detect melanoma cells in two of eight patients (25%).

Pre-operative staging of cervical cancer: comparison of magnetic resonance imaging (MRI) and computed tomography (CT) with histologic results.

Thirty-two patients with histologically confirmed cervical carcinoma were preoperatively investigated using MRI; in addition, a CAT-scan was performed on 15 of these patients. The diagnostic results using both modalities were compared with the histological findings (after hysterectomy according to Wertheim-Meigs, including lymph node dissection in the pelvic and, in part, in para-aortal regions). Determination of tumour volume was possible with high accuracy using MRI. Accuracy in assessing the parametria was 86%, vagina 90%, bladder and rectum 97%. The shortcoming of MRI is still the detection of infiltrated lymph nodes. The accuracy of 69% achieved for lymph nodes is equal to results with computed tomography. The general accuracy for our patients in staging was 81% for MRI versus 47% for CT. MRI-based diagnosis enables us to determine a correct tumour staging preoperatively, and is therefore very helpful in planning an adequate therapy. If MRI were used more widely it would contribute to simplification and shortening of the preoperative diagnostic procedure in patients with cervical carcinoma.

1H and 31P NMR characterisation of a double breast coil for spectroscopic measurements and imaging.

For the first time a double turn breast coil has been described which can be used for 1H imaging, 1H spectroscopy and 31P spectroscopy. The paper describes basic technical features of the coil, coil design, B1 field/excitation field distribution for 1H and 31P, sensitivity, and feasibility for 31P spectroscopic in vivo studies. The main advantage of the double frequency tuneable coil is that 1H imaging for tumor localization and 31P spectroscopy for response control can be done without an additional repositioning of the patient.

Fluoride release/uptake of polyacid-modified resin composites (compomers) in neutral and acidic buffer solutions.

The aim of the present study was to evaluate the fluoride uptake/release of polyacid-modified resin composites (compomers) in neutral and acidic buffer solutions. Two compomers (Dyract and Compoglass) were tested and the conventional glass-ionomer cement (GIC) Vivaglass Base served as a control. Forty specimens were fabricated from each of the respective materials. Twenty of these specimens were placed in artificial saliva and the other 20 specimens in a fluoridated dentifrice slurry for 5 min. Then, 10 fluoridated and 10 non-fluoridated specimens were immersed in a neutral buffer solution (pH 6.8), and the other specimens were immersed in an acidic solution (pH 4.0). After 1, 2, 3, 4, and 5 days the samples were again placed in either a fluoridated dentifrice slurry or saliva for 5 min, after which time they were transferred to fresh buffer solutions. The fluoride content of the solutions was assessed with a fluoride sensitive electrode. Fluoride release from all the materials decreased continuously during the experiment, with a significantly higher release in the acidic solution compared to the neutral buffer solution. Fluoridation did not result in an increased fluoride release for the compomers. However, the conventional GIC revealed a significantly higher fluoride release after fluoridation. It is concluded that Dyract and Compoglass can not be replenished with fluoride, irrespective of the pH value of the environment.

[Effects of para- and diamagnetic materials in a 1.5 Tesla high field magnetic resonance tomography system]. / Effekte von para- und diamagnetischen Materialien in einem 1,5-Tesla-Hochfeld-Magnetresonanztomographen (MRT).

This article shows the results of an experimental investigation of the interference by paramagnetic and diamagnetic materials on imaging in a closed 1.5 Tesla high field magnetic resonance imaging system (MRI). For different types of sequences (SE, GE, EPI) the effects of metal and non-metal profiles in producing artefacts were investigated. A phantom (plastic trunk) filled with Gd-Mn-solution was used for representation of the artefacts. The materials analysed were placed parallel to the phantom at predetermined distances. The images were obtained in transverse and sagittal planes and analysed with respect to the resulting artefacts. The results show that aluminum and polymer profiles produce the weakest artefacts, even when the material is positioned close to the phantom. A comparison of the sequence types shows that the SE-sequence has a low sensitivity to artefacts, despite the great profile variation in size and shape. The SE-sequence accordingly showed a higher imaging stability as compared with the GE- and EPI-sequences. Steel and copper produced the strongest artefacts. The examination was begun after an intensive study of the literature (Internet, Medline, Meditec). So far have been few publications on this subject.

[Cervix carcinoma: staging, therapy, after-care. Experiences with magnetic resonance tomography of cervix carcinoma based on recent literature 1993 to 1997]. / Zervixkarzinom: Staging, Therapie, Nachsorge. Die Erfahrungen in der Magnetresonanztomographie des Zervixkarzinoms anhand der neuen Literatur von 1993 bis 1997.

The investigation of cervix carcinoma with magnetic resonance tomography (MRT) is still controversially discussed with regard to its diagnostic value as well as for planning radiation therapy. The purpose of this article is to present and discuss papers published between 1993 and 1997 in this field with respect to the technique used, the contrast media applied and its clinical value. A literature search using three different databases (Medline, Embase, Cancerlit) identified 39 publications, which were then analysed. Despite the partially suboptimal presentation of results in these papers MRT proved superior to other imaging modalities. Due to better demarcation of cervix carcinoma with MRT, it was possible to calculate tumor value as well as to correctly judge the infiltrative character. This allows for a more precise treatment and staging of the patient's prognosis. In the future, MRT might be useful in diagnosing recurrence at relatively early stage. Unfortunately lymphatic nodes can only be insufficiently verified using MRT.

[Imaging tumor extension of renal cell carcinomas with magnetic resonance tomography. Improved tumor-tissue contrast with Gd-DTPA-assisted spin-echo sequences and simultaneous fat suppression]. / Darstellung der Tumorausdehnung von Nierenzellkarzinomen mit der Magnetresonanztomographie. Verbesserung des Tumor-Gewebekontrastes mittels Gd-DTPA unterstützter Spin-Echo-Sequenzen und gleichzeitiger Fettsättigung.

PURPOSE: To evaluate the use of contrast-enhanced T1-weighted images with fat suppression (T1FS) to improve the contrast-to-noise ratio of renal cancer and renal parenchyma as well as perirenal fat. METHODS: 25 patients with histologically proven unilateral renal cancer after nephrectomy were examined before surgery. In addition to plane and contrast-enhanced T1-weighted as well as T2-weighted spin-echo images, all patients had T1 FS immediately after administration of Gd-DTPA in two planes. The contrast-to-noise ratio was calculated using circular regions-of-interest which outlined the tumor, the renal parenchyma, pyelon, and the perirenal fat. RESULTS: T1 FS significantly improved the contrast-to-noise ratio of renal cancer and renal parenchyma compared to all conventional spinecho sequences (p < 0.001; Wilcoxon-Test). Compared to contrast-enhanced T1-weighted images without fat-suppression T1 FS yielded a higher CNR of the tumor, the perirenal fat and the pyelon. Another advantage was the absence of the chemical-shift artifact which is mostly pronounced in T2-weighted images and a reduced number of observed artifacts due to breathing. CONCLUSION: T1 FS should replace conventional contrast-enhanced T1-weighted spin-echo images in the work-up of renal cancer using MRI.

Immunomagnetic enrichment of disseminated epithelial tumor cells from peripheral blood by MACS.

Disseminated epithelial tumor cells have been detected in the bone marrow and blood of cancer patients by means of immunocytochemical or immunofluorescent staining of cytocentrifuge slides, multiparameter flow cytometry, and reverse transcriptase-polymerase chain reaction. However, it is hardly possible using such methods to detect tumor cells at a frequency below 10(-6). To increase the sensitivity of these detection techniques we have developed a new technology for the enrichment of disseminated epithelial tumor cells from hematopoietic cell samples by high-gradient magnetic cell sorting (MACS). Cells are permeabilized and fixed and carcinoma cells are magnetically labeled specifically with an anti-cytokeratin 8 monoclonal antibody (mAb) directly conjugated to superparamagnetic microbeads. Magnetically labeled cells are enriched on high-gradient magnetic columns. Tumor cells are detected in the enriched cell fraction by flow cytometry, fluorescence microscopy, or immunocytochemisty. In this study we demonstrated the method using a model system in which five to 5,000 cells from a breast cancer cell line were seeded into blood cell samples from a healthy donor containing 1.2 x 10(8) leukocytes. Tumor cells were 10,477+/-4242 (n=25)-fold magnetically enriched, and 57.7%+/-16.9% (n=33) of the initially seeded tumor cells were recovered. Applying the method to 20-40 mL blood samples from patients with advanced carcinomas of the breast, prostate, colon, rectum, or lung, we were able to detect between one and 6.8 x 10(4) cytokeratin-expressing tumor cells in 21 of 34 patients. This corresponds to frequencies of tumor cells between 6.8 x 10(-9) and 1.1 x 10(-3) among nucleated cells in the original sample. Enriched tumor cells were further analyzed for expression of tissue-specific and prognostic markers such as breast mucin glycoproteins, erbB2, and CD44v6 for additional characterization and to confirm their tumor origin. The technique described could become a valuable tool for the quantification and molecular characterization of metastatic carcinoma cells in hematopoietic tissue, and may ultimately prove useful in the diagnosis, prognosis, and monitoring of patients with carcinoma.

[Optimal use of MRI mammography from the economic viewpoint]. / Der optimale Einsatz der MR-Mammographie (MRM) unter wirtschaftlichen Gesichtspunkten.

With the introduction of the contrast agent gadolinum DTPA there were hopes that "MRM" would prove to be the investigatory technique that would largely solve the problems of breast diagnostics. However, after the early years of acceptance, the new method of investigation became a subject of controversy. Nonetheless, MRM today occupies a recognized place in diagnostics for certain indications. It is still true, however, that reliable use of this procedure requires a great deal of experience, since there is a relatively large area of overlap between benign and malignant tumors. Further, the costs are significantly higher than those for conventional methods of investigation. New studies that have been conducted at the Charité, Campus Virchow Medical Center in Berlin, suggest that, if one takes the relevant indications into account, MRM can be economic and contribute significantly to cost reduction. Application of a newly developed software package has shown that the good discrimination in a suspect area resulting from contrast agent enhancement makes possible a reliable differentiation between malignant and benign tissue changes. A further result was that, when certain boundary conditions are satisfied, a contrast agent bolus of 0.1 mmol/kg BW is sufficient, making a double dose (0.2 mmol/kg BW) unnecessary.

Skeletal muscle-specific myosin binding protein-H is expressed in Purkinje fibers of the cardiac conduction system.

Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5' regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of approximately 86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.

Boeck sarcoidosis of the breast: mammographic, ultrasound, and MR findings.

The rare case of sarcoidosis of the breast is presented. The mammographic, ultrasound, and MR appearances are described.

Is there a correlation in breast carcinomas between tumor size and number of tumor vessels detected by gadolinium-enhanced magnetic resonance mammography?

Tumor vessels are known as a sign of malignancy in breast tumors. Is there a correlation between tumor size and the number of vessels in cases of breast tumor examined by dynamic gadolinium (Gd)-enhanced MR imaging? Eighteen patients (mean age, 46 +/- 7 years) underwent dynamic Gd-enhanced MR imaging of the breast by three-dimensional gradient echo sequence using thin-layer technique (2.5 mm) at 1.5T. The dynamic study included one precontrast and four postcontrast sequences (every 90 seconds) in coronal slices. Postprocessing by subtraction method and reconstruction in both transverse and sagittal planes were performed. All carcinomas showed rapid Gd enhancement. Tumor size (0.5 to 31.5 cm3; mean, 6.3 +/- 3.7 cm3) and number of vessels (1 to 10; mean, 3 +/- 2.1) were detected in summation of all three directions. A significant correlation was found between number of vessels and tumor size (r = 0.787, p < or = 0.01). Breast tumor size significantly correlated with the number of vessels detected by Gd-enhanced MR mammography. The introduced method is a further important step in differentiating a carcinoma from a benign lesion.