[Genetic methods for analysis of autoinflammatory diseases]. / Genetische Methoden für die Analyse autoinflammatorischer Erkrankungen.

Over the past years the phenotypic and genetic spectrum of autoinflammatory diseases has continuously increased. Moreover, several monogenic autoinflammatory disorders have now been identified where febrile episodes are not among the leading symptoms and which can be accompanied by autoimmune phenomena and susceptibility to infections. Autoinflammatory conditions that are characterized by uncontrolled activity of cytokines, such as interleukin-1 beta (IL1ß), tumor necrosis factor alpha (TNF-α) and type 1 interferons (1-IFN), are amenable to specific therapeutic interventions. Thus, identification of the underlying genetic cause is important. During diagnostic work-up, genetic testing of a patient with autoinflammation should be carried out depending on the clinical presentation. If a distinct disorder is suspected, sequencing of the causative gene should be performed. Genetic tests using next generation sequencing (NGS), such as panel sequencing, exome sequencing and array comparative genomic hybridization (CGH) can be carried out if symptoms cannot be assigned to a specific disease entity.

Bimodal endoscopic probe combining white-light microscopy and optical coherence tomography.

We present a novel bimodal endoscopic imaging probe that can simultaneously provide full-field white-light video microscopy and confocal optical coherence tomography (OCT) depth scans. The two modalities rely on spectrally separated optical paths that run partially in parallel through a micro-optical bench system, which has a cross-section of only 2 mm×2.76 mm and is realized via standard silicon micromachining techniques. With a numerical aperture of 0.061, the video modality has a resolution and field of view of 9.3 and 1240 µm×1080 µm, respectively. The resolution is limited by the pixel spacing of the coherent fiber bundle, which relays the acquired image from the distal to the proximal end. A custom-designed diffractive optical element placed within the video imaging path significantly improves the image contrast by up to 45% in the medium frequency range. The OCT modality is optimized for 830 nm center wavelength, and works in a confocal arrangement with an NA of 0.018. It provides single-point depth probing at the center of the video image with a lateral resolution of 20 µm. Through its compact footprint and enhanced functionality, the probe can provide depth-resolved guiding capability for existing laparoscopes and represents a major step toward a new class of multimodal endoscopic imaging probes.

Tailoring the optical properties of atomically-thin WS2via ion irradiation.

Two-dimensional transition metal dichalcogenides (TMDCs) exhibit excellent optoelectronic properties. However, the large band gaps in many semiconducting TMDCs make optical absorption in the near-infrared (NIR) wavelength regime impossible, which prevents applications of these materials in optical communications. In this work, we demonstrate that Ar+ ion irradiation is a powerful post-synthesis technique to tailor the optical properties of the semiconducting tungsten disulfide (WS2) by creating S-vacancies and thus controlling material stoichiometry. First-principles calculations reveal that the S-vacancies give rise to deep states in the band gap, which determine the NIR optical absorption of the WS2 monolayer. As the density of the S-vacancies increases, the enhanced NIR linear and saturable absorption of WS2 is observed, which is explained by the results of first-principles calculations. We further demonstrate that by using the irradiated WS2 as a saturable absorber in a waveguide system, the passively Q-switched laser operations can be optimized, thus opening new avenues for tailoring the optical response of TMDCs by defect-engineering through ion irradiation.

Posttraumatic aneurysm of the right atrium.

We report on an acquired right atrial false aneurysm, which was removed under extracorporeal circulation. The patient remembered three occasions of blunt chest trauma with rib fractures. Clinical symptoms were ongoing dyspnea, chest pain, and atrial fibrillation.

Detection of myocardial viability by low-dose dobutamine Cine MR imaging.

The purpose of this work was to test the diagnostic value of dobutamine stress magnetic resonance imaging (MRI) for predicting recovery of regional myocardial contractility after revascularization. Cardiac wall motion abnormalities are due to either non-viable and/or scarred, or viable, but hibernating, myocardial tissue. Dobutamine stress leads to increased systolic wall thickening only in viable myocardium. Twenty-five patients with akinetic or dyskinetic myocardial regions were examined with a Cine FLASH-2D sequence at rest and during dobutamine stress (10 microg/kg/min). Patients were re-examined at rest 3, and in case of persisting wall motion defects, 6 months after revascularization. Criterion of viability was increasing end-systolic wall thickening during stress and/or at follow-up. Akinetic regions related either to the LAD (n = 19) or to the RCA (n = 6) were judged viable if > or = 50% of the affected segments improved. MR studies were completed in all subjects without arrhythmia or need for early terminations due to symptoms. Sensitivity, specificity, and positive predictive value for the prediction of myocardial viability were 61%, 90%, and 87% for the segment-related analysis, and 76%, 100%, and 100% for the patient-related analysis based on coronary artery distribution, respectively. Dobutamine stress MRI allows to predict global functional recovery of akinetic myocardial regions after revascularization with a high positive predictive value and high specificity.

[A comparison of Ultrax, Diazil, Bactrim and Spiramycin in experimental toxoplasmosis in mice (author's transl)]. / Vergleichende Untersuchung der Wirksamkeit von Ultrax, Diazil, Baktrim und Spiramycin auf die experimentelle Toxoplasmose der Maus.

White mice infected intraperitoneally with the RH-strain of Toxoplasma gondii (inoculum size 50,000 to 100,000 free protozoans per mouse) received treatment between the second and eighth day after infection with sulphamethazine-pyrimethamine, sulphamethoxy-diazine-pyrimethamine, trimethoprim-sulphamethoxazole or spiramycin subcutaneously. All untreated controls and all mice of the trimethoprim-sulphamethoxazole and spiramycin-treated groups died during the acute stage (except two mice in the latter group on the 15th day). The mean survival times were 6.5, 7.5 and 7.6 days, respectively. The best results were obtained in the sulphamethazine-pyrimethamine-treated mice; 18 out of 27 survived the 30-day observation period (5 cured), in contrast to only 9 out of 40 sulphamethoxydiazine-pyrimethamine-treated mice. Considering the high pathogenicity of the RH-strain, the differences in immunological defence mechanisms in mice (absence of antibody-activating "accessory factor") and the late commencement of treatment of the infected mice, one can state that the combination of sulphamethoxydiazine-pyrimethamine should also be capable of overcoming acute human toxoplasmosis in pregnancy. Spiramycin, by contrast, should be given only in cases where sulphonamide intolerance exists and must then be given in high doses until delivery. The combination of sulphamethoxazole-trimethoprim cannot be recommended.

Alcohol acutely increases vascular reactivity together with insulin sensitivity in type 2 diabetic men.

Moderate alcohol consumption is associated with increased insulin sensitivity and reduced cardiovascular risk. We hypothesized that this relates to a direct effect of alcohol and therefore investigated whether acute alcohol intake altered insulin sensitivity or endothelial function in patients with type 2 diabetes. In an open-label two period design, the effect of a single oral dose of 40 g of alcohol (168 ml 40% vodka) on an insulin-modified frequently sampled intravenous glucose tolerance test (FSIGT) and on endothelium-dependent (flow mediated, FMD) or endothelium-independent (glyceroltrinitrate (GTN)-induced) vasodilation of the brachial artery measured by ultrasound was studied. Experiments were carried out in twelve male patients with type 2 diabetes mellitus (64+/-6 years, body mass index 28.4+/-5.7 kg/m (2)). Baseline insulin sensitivity index (S (I)) was 1.10+/-0.34 min (-1).microU (-1).ml, baseline FMD was +4.1+/-3.0%, and GTN-induced vasodilation +7.4+/-2.3% from resting brachial artery diameter. Acute alcohol intake increased alcohol plasma levels to 0.33+/-0.04 per thousand, S (I) to 1.86+/-0.45 min (-1).microU (-1).ml (p<0.05), and FMD to +8.2+/-2.8% (p<0.05), while GTN-induced dilation remained unchanged. No relationship was detectable between the observed changes. We conclude that alcohol intake acutely increases endothelium-dependent brachial artery vasodilation in patients with type 2 diabetes together with insulin sensitivity. This acute effect might explain some beneficial effects of low alcohol consumption in epidemiological observations.

Kinetics of vegetative growth of Thermoactinomyces vulgaris.

The growth of Thermoactinomyces vulgaris on solid media was studied by microculture technique. Starting from a germinating spore the further development was followed by phase contrast microscopy. From serially taken photographs the kinetics of growth and branching of the mycelium was calculated. Also some observations on morphological behaviour were made. Regarding the total mycelium developing from one spore the growth occurred witha constant exponential growth rate, at least for the time tested (about ten doublings). The constancy of the mycelial growth rate was not attributed to constant rates of hyphal growth or branching. The hyphal growth rate increased during the development of the mycelium, so that the length produced by a hypha within a given time was smallest after germination and largest at the end of the experiment. Between the first and the 10th doubling of the mycelium the mean hyphal growth rate increased by the factor 27, in some cultures even by the factor 40. While hyphal growth changed from exponential to linear, branching was first linear and then approached exponential kinetics. Under different conditions of cultivation mycelia could have identical doubling times, but different hyphal growth rates and vice versa. Yeast extract caused a reduction of the hyphal growth rate and an enhancement of branching. Likewise the diminution of the supply of oxygen did not reduce the mycelial growth rate, but markedly decreased the hyphal growth rate, especially of the germ tube. The data obtained concerning the kinetics of growth and branching can be explained by a special form of cooperation of cytoplasmic and cell wall synthesis. With respect to the synthesis of cytoplasma a T. vulgaris mycelium behaves as one individual, which is not differentiated into replicating and non replicating areas (as recently was shown to be likely for Streptomyces hygroscopicus by Schuhmann and Bergter 1976). The mycelial growth rate thus reflects the rate of the synthesis of cytoplasm. The hyphal growth rate is moreover determined by the rate of cell wall synthesis at the tip of each hypha. The observed increase of the hyphal growth rate is explainable by an acceleration of the cell wall synthesis during the development of the mycelium. Probably the branching is regulated indirectly. According to the different manner of cytoplasmic and cell wall synthesis, within a hypha a balance between both processes is maintainable only for a limited time. As soon as the momentary apical capacity for cell wall synthesis is exhausted, the balance is attained anew by originating a new site for wall synthesis, i.e. by branching. Branching was often defective. Especially in the absence of yeast extract a lot of lateral branches arose, which ceased further growth at an average length of 2.5 micron. It is suggested that yeast extract supports the transport of the nucleoids into the lateral branches, which otherwise is often infeasible. Reduction of the supply of oxygen favoured apical branching, so that dichotomous forms resulted.

Dependence of the mycelial growth pattern on the individually regulated cell cycle in Streptomyces granaticolor.

The growth behaviour of Streptomyces granaticolor ETH 7437 was studied by the microculture technique. The kinetics of growth and branching were recorded and, since elongation was found to be restricted to apical elongation sites (e-sites), the rate of elongation per site (alpha) was determined as well. The mycelia grew exponentially. Initially the growth was dependent on alpha of the germ tube, but after the start of branching, growth paralleled the exponential increase of the number of branches while alpha attained a constant average value. Further, for liquid grown mycelia showing about the same growth kinetics the cellular structure was determined after cell wall staining. Three types of cells could be distinguished: apical branchless cells (20%), non-apical branchless cells (20%) and non-apical cells with one branch each (60%). Since both the apical and the branched cells possessed an e-site, 80% of the cells must have been growing at the time of sampling. Combining detailed data obtained from both the alive and the stained mycelia a model was elaborated, which may reflect the events taking place on the cellular level during mycelial growth. The model is based on the assumption that each cell behaves as an independent unity with respect to its cell cycle. But, in contrast to the behaviour of single cell bacteria, in mycelia the two daughter cells formed upon division are neither equivalent nor uniform. Here, the sister cells differ in length, shape and possession of an e-site. Only one of the daughter cells receives the e-site of the mother cell, while the other starts its own cell cycle by generating a new e-site at the cylindrical part of its envelope. Regarding the length of sister cells the degree of heterogeneity increases with the age of the corresponding region of the mycelium, and eventually some cells lose the ability to generate an e-site, i. e. to grow. With this model the kinetic and structural peculiarities of the mycelial growth of Streptomyces granaticolor can be explained.

Localization of alkaline phosphatase in Streptomyces noursei.

Alkaline phosphatase activity appeared in Streptomyces noursei strain IMET 43,716 when cultures were shifted to phosphate limitation. By using p-nitrophenylphosphate as substrate, the activity detected at pH 9.4 was cell-bound, as long as young mycelia were studied. Supernatant fluids of the cultures were only active, if partial mycelial lysis had taken place after incubation for several days under the influence of shear forces. After cytochemical staining the phosphatase reaction products were microscopically visible as grains distributed rather homogeneously within the hyphal lumen. The frequency of grains was correlated to the rate of nourseothricin production. Electron microscopy of thin sections showed the phosphatase reaction products to be only present in the cytoplasm.

Morphogenetic behaviour of two Streptomyces strains analyzed by the use of chemostats.

Streptomyces granaticolor ETH 7347 and Streptomyces hygroscopicus IMET JA 6599 were cultivated in Casamino acids-limited chemostats. Amino acids served as sole source of energy, carbon and nitrogen, but their limitation affected primarily the catabolism of the mycelia. At different specific growth rates (mu) samples were withdrawn. After staining the cell walls of the mycelia the average length of the cells (mycelial length per cross wall: L/C) and of the hyphal growth units (mycelia-length per branch: L/N) were determined. L/C was only slightly influenced by mu. In S. granaticolor the cells were on average 11.5 microns and in S. hygroscopicus about 18.5 microns long. However, L/N was dependent on mu. Upon a shift from mu = 0.6 to mu = 0.1 L/N was reduced 1.2 fold in S. granaticolor and 1.8 fold in S. hygroscopicus. Especially growth rates below 0.2 stimulated branching. The opposite response of L/N to changes of mu was, however, observed in glucose-limited chemostat cultures (Riesenberg and Bergter 1979). Thus, the average length of the growth units is not determined by mu per se--even if in both cases the energy source was limited--but by specificities of the nutrient-dependent metabolism. Comparison of L/C and L/N showed that in all samples of S. granaticolor cells were smaller than the hyphal growth units, while they were larger in S. hygroscopicus. From a cellular point of view, thus there exist different mechanisms of branching--the single and the multiple branching--which were discussed.

Analysis of morphogenesis of the nocardioform organism Oerskovia xanthineolytica.

Depending on the nutritional and physicochemical conditions of growth the shape of Oerskovia xanthineolytica varied within a broad range. In different exponentially growing cultures five morphological types could be distinguished. In liquid cultures with increasing growth rate Oerskovia grew either as rods, filaments or branched filaments, whereas for the agar-microcultures pseudomycelia, but under reduced aeration mycelia were typical. The morphogenetic parameters of each type were determined, such as frequencies of septation and cell separation and - since wall extension was found to occur by the synthesizing activity of elongation sites (e-sites), their frequency, position and elongation rate as well. The cell length varied between 1 and about 20 micron, roughly correlated to the specific growth rate, but was also influenced by the composition and the consistence of the medium. The longest cells were found within the faster growing cultures, forming branched filaments, mycelia or pseudomycelia. During transition to the stationary growth phase these forms fragmented into rods by increase of the frequency of septation and cell separation. Increased cell length was accompanied by a reduced frequency of e-site formation which was compensated by an enhancement of their synthesizing activity. The rate of envelope synthesis varied with the morphological type from 0.12 to 9.60 micron/h. In agar-microcultures these values were much higher than in liquid media. In liquid cultures the e-sites preferentially were situated at one (rod) or the 2 cell poles (filaments), but during faster growth additional e-sites were formed within the cylindrical part of the envelope, thus leading to branching. In pseudomycelia the e-sites were formed laterally at the poles. In mycelia the poles did not receive e-site-activity, which instead occurred remote from the cell poles, also causing branching. This means that branching is either the result of the formation of more than two (up to 7) e-sites per cell (fast growing liquid cultures) or of a specific lack of transforming the poles into e-sites, (weakly aerated agar-cultures). The separation of sister cells was correlated to the transformation of poles into e-sites.

Alteration of interaction with virulent bacteriophage Ta1 during differentiation of Thermoactinomyces vulgaris.

During its life cycle Thermoactinomyces vulgaris 1227 and 1261 substrate mycelium showed three modes of interaction with the virulent phage Ta1, each expressed at a distinct morphological stage. Primary mycelium arising from spores 50 min after start of germination was the only stage which propagated phages upon infection. However, infection of growing secondary mycelium or of late sporulation stages resulted in a loss of phage. The lack of phage production upon infection of growing secondary mycelium was not related to sporogenesis, since it appeared already 3.5 hours before the beginning of sporulation. If phages were added to the secondary mycelium at the beginning of spore formation, the phage genome became integrated in the developing spores in a heat-stable state. Allowing outgrowth of these prophage-carrier spores, phages were produced similarly as in germ tubes arising from normal spores infected at the time of inoculation. The growing secondary substrate mycelium was characterized by competence for the uptake of exogenous DNA. Since at the stage of competence phages were neither produced nor was phage DNA trapped, the earlier reported lack of transfection in undisturbed differentiating T. vulgaris is now understandable.

Location of branches within the apical hyphal region of Streptomyces granaticolor mycelia.

The location of branches (ramifications) within the apical hyphal region of Streptomyces granaticolor mycelia was studied in dependence on the specific growth rate by using batch and chemostat cultures. The site where a new branch emerged was correlated to the neighboured elongation sites (e-sites) as well as to the septae. In dependence on the growth rate the distance from a newly formed branch (i.e. e-site) to the apical e-site (tip of the main hypha) changed from 12 to 44 microns and to the e-site on the tip of the preceding neighboured branch from 12 to 27 microns. Thus, except for the slowest grown culture, the new branching site in the apical hyphal region did not represent the midst between the two preexisting e-sites. Comparing branching and septation sites, the data indicated a close correlation between them, if the respective mean values of whole populations were determined. But regarding individual hyphae, only 4-39%--depending on the growth rate--of the apical branches were directly neighboured to a septum. The remaining percentage was situated at one of several potential branching sites, the number of which per subapical cell corresponded to the number of nucleoids.

[Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]. / Einbau von Thymidin in die DNS von Actinomyceten. I. Der Einbau exogenen Thymidins in die DNS von Thermoactinomyces vulgaris.

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.

DNA replication is not restricted to specific regions in young vegetative Streptomyces mycelia.

In order to determine the localization of DNA-synthesis in Streptomyces granaticolor and Streptomyces hygroscopicus, mycelia (growing either on agar or in liquid medium) were pulse-labelled with 3H-thymidine and prepared for autoradiography. The distribution of silver grains showed no regions of preferential incorporation of 3H-thymidine in mycelia up 300 micron in length. Since mycelia grow by apical elongation of hyphae, the frequency of silver grains was quantitatively analysed along individual main hyphase. No significant difference of labelling was found within zones of different age up to a distance of 80 micron from the hyphal tip. Also, the very youngest part of the hyphae enclosing only the most apically situated nucleoid did not show any deviation from the average frequency of silver grains.

Autolysis of Thermoactinomyces vulgaris spores lacking carbon dioxide during germination.

Ultrathin sections of early germinating endospores of Thermoactinomyces vulgaris were studied by electron microscope. Only spores aerated with an air-CO2 mixture (5% CO2) grow out, while spores aerated with air (0.03% CO2) lyse by the 25th min of inoculation. The lysis is due to progressive, unlimited degradation of the spore integuments and a lack of cell wall formation around the spore protoplast. The requirement of CO2 for outgrowth could not be replaced by oxaloacetate. CO2 seems to be needed to energize the dormant cytoplasmic membrane of the spore to render it capable of initiating active transport processes and of synthesizing the germ cell wall.

Properties of Thermoactinomyces vulgaris phage Ta1 and its extracted DNA.

The virulent phage Ta1 was obtained in good yields from infected cultures of Thermoactinomyces vulgaris 1227. The purified phage was found to sediment with a single band, the sedimentation constant being (519 +/- 14)S, and to exhibit a typical nucleoprotein behaviour in UV-spectrophotometric and CD experiments. The Ta1 phage consists of a hexagonal head about 0.056 micrometers in diameter and a very short tail. It is morphologically similar to the temperate Salmonella phage P22. The nucleic acid extracted from the phage was found to be a double-stranded linear DNA with a G+C content of 42 mole-% as deduced both from its melting temperature and buoyant density in CsCl. Analytical sedimentation revealed a high degree of molecular homogeneity of Ta1 Dna. the sedimentation constant of this DNA amounts to (35.9 +/- 0.3)S, corresponding to a DNA molecular weight of about 29 millions daltons. The biological activity of Ta1 DNA was indicated by its ability to infect the mycelium of the components T. vulgaris strain 1227 and to give rise to mature phages.

Interaction of bacteriophage O2 with strains of the genus Oerskovia.

Bacteriophage O2 multiplies normally on Oerskovia turbata IMET 47 153. It has a burst size of about 100 p.f.u. per infected cell and a latent period of 100 min at 30 degrees C. On Oerskovia xanthineolytica IMET 47 383 clear spots were formed after addition of high phage concentrations onto agar top layers. By phase contrast observation, and measurement of the optical density of infected cultures, it was found that the clearing effect on strain IMET 47 383 was due to lysis-from-without. Phage O2 adsorbs and injects its DNA into cells of strain IMET 47 383 but phage multiplication does not occur, and the phage DNA becomes degraded. Inhibition of phage DNA injection by the combined action of xanthotoxin -- u.v. irradiation abolished the clearing activity of phage lysates. Therefore, both adsorption and DNA injection seem to be prerequisites for the release of a lytic activity out of the phage particle, which is responsible for the clearing effect on strain IMET 47 383.

[Post-traumatic aneurysma spurium of the right atrium]. / Posttraumatisches Aneurysma spurium des rechten Vorhofes.

In a 61 year old male with heart failure and pulmonary congestion the x-ray shows a right paracardial tumor. The patient suffered from a blunt chest trauma 6 years ago. Since that accident he complains about exercise related dyspnea and cardiac arrhythmia with atrial fibrillation. On echocardiography we found a echolucent cystic tumor with a solid center structure surrounded by a thin membrane. Doppler echocardiography revealed a heart cycle dependent flow at its margin. During dextrocardiography rapid opacification only of the peripheral structures of the tumor could be observed. These findings are consistent with a traumatic rupture of the right atrium, and the diagnosis of a posttraumatic aneurysma spurium of the right atrium was established. Surgery confirmed this diagnosis and the aneurysm was extirpated.