Immunostimulatory DNA: sequence-dependent production of potentially harmful or useful cytokines

Certain bacterial immunostimulatory (i.s.) DNA sequences containing unmethylated CpG motifs stimulate antigen-presenting cells (APC) to express a full complement of costimulatory molecules and to produce cytokines including interleukin (IL)-12 and tumor necrosis factor (TNF)-alpha. While IL-12 is key to their T helper cell (Th)1-promoting adjuvant activity, secretion of toxic levels of TNF-alpha is harmful in that it promotes toxic shock. Given the beneficial as well as harmful consequences of i.s. DNA, we investigated the possibility of identifying DNA sequences, i.e. CpG oligodeoxynucleotides (ODN) which differentially activate IL-12 versus TNF-alpha cytokine production in APC. Here, we describe an i.s. DNA sequence with these characteristics. While its potential to induce IL-12 is preserved, its ability to trigger TNF-alpha release is strongly curtailed both in vitro and in vivo. I.s. DNA could be segregated into lethal and non-lethal in a mouse toxic shock model. The non-toxic i.s. DNA was useful as an adjuvant, thus allowing cytotoxic T cell responses to the soluble protein ovalbumin and conferring a resistant Th 1 phenotype to BALB/c mice lethally infected with Leishmania major. This i.s. CpG motif may thus be prototypic for a useful immunostimulating DNA sequence that lacks harmful side effects.     

Detection of Staphylococcus aureus enterotoxins A to D by real-time fluorescence PCR assay

Staphylococcus aureus is one of the most significant pathogens causing nosocomial and community-acquired infections. Among the secreted staphylococcal virulence factors, there is a growing list of enterotoxins which can induce gastroenteric syndrome and toxic shock syndrome. Here, we developed a real-time fluorescence PCR assay (TaqMan PCR) for the detection of genes encoding staphylococcal enterotoxins A, B, C1, and D (SEA, SEB, SEC1, and SED) of S. aureus as well as the mecA gene encoding methicillin resistance and the femB gene as a specific genomic marker for S. aureus. SEA to SED were selected because they are the four classically described enterotoxins of S. aureus and because they were detected by latex agglutination. In order to evaluate the reliability of TaqMan PCR, we investigated 93 isolates of S. aureus derived from patients at our hospital over 5 months and compared the results with data obtained by a commercially available reversed passive latex agglutination assay (SET-RPLA) for these isolates. Thirteen enterotoxin genes were detected by TaqMan PCR; however, no proteins expressed by these genes were detected by SET-RPLA. As a result, more isolates of S. aureus (n = 44) were found positive by TaqMan PCR for one or more enterotoxin genes than by SET-RPLA for the respective proteins expressed by these genes (n = 40). We conclude that TaqMan PCR is more sensitive because it offers the possibility for determining enterotoxins on a genotypic basis. Additionally, the assay allows the parallel detection of genes for SEA to SED and methicillin resistance in S. aureus. Furthermore, real-time PCR is well suited for screening large numbers of samples at the same time, allowing rapid, reliable, efficient, and cost-saving routine laboratory diagnosis.     

Increased susceptibility to postoperative sepsis in patients with impaired monocyte IL-12 production

IL-12 is a potent immunoregulatory cytokine that is essential for the development of protective immunity, as demonstrated by numerous animal models of infection. Here, we provide evidence for a critical role of IL-12 in human sepsis. The results of a prospective study of 184 patients undergoing major elective surgery of the upper and lower gastrointestinal tract revealed that, in contrast to patients showing uneventful recovery, monocyte IL-12 production was severely and selectively impaired in patients developing postoperative sepsis. Moreover, the extent of monocyte IL-12 suppression correlated with the severity of postoperative sepsis. Monocyte IL-12 secretion was suppressed before surgery and remained low until the onset of sepsis. Therefore, the suppression of IL-12 secretion preceded the onset of postoperative sepsis but did not occur as a consequence of major surgery. In contrast, IL-1beta production was only reduced during the late postoperative course in patients developing postoperative sepsis, and TNF-alpha release was even increased at different time intervals before the onset of sepsis. Thus, reduced IL-12 release does not reflect a general defect in monocyte cytokine production. Consequently, these results establish a critical role for IL-12 in early resistance to postoperative infection and may allow for the development of novel therapeutic strategies designed to stimulate host defense mechanisms and to reduce the incidence and severity of septic complications.     

Simultaneous occurrence of a supra- and an infratentorial glioma in a patient with Ollier's disease: more evidence for non-mesodermal tumor predisposition in multiple enchondromatosis

A case is presented in which two neuro-ectodermal tumors, an infra- and a supratentorial glioma, developed in a young man with multiple enchondromatosis of Ollier's disease. This is the third such case of multifocal low-grade glioma in Ollier's disease, suggesting a predisposition for non-mesodermal tumors in Ollier's disease. The related condition of multiple enchondromatosis and hemangiomas (Maffucci's syndrome) is well known for its malignant potential, developing both mesodermal and non-mesodermal tumors. Along with other authors, we support the concept of two variants of the same disease with a predisposition to development of tumors from various germ layers.     

HRTEM investigation of the epitaxial growth of scandate/titanate multilayers

Despite their potential application in strain-engineered layer structures, the epitaxial growth of rare earth scandate films on cubic perovskite substrates is not fully understood to date. Here we report on the epitaxy of the orthorhombic GdScO₃ and DyScO₃ on (001) BaTiO₃ and SrTiO₃ thin films. In particular, the orientation relationship between the orthorhombic epilayer and the cubic substrate is examined by aberration corrected high-resolution transmission electron microscopy. For all cases investigated, the long axis of the orthorhombic unit cell was found to be perpendicular to the growth direction.     

Evaluation of the pore morphology formation of the Freeze Foaming process by in situ computed tomography

The so-called Freeze Foaming method aims at manufacturing ceramic cellular scaffolds for diverse applications. One application is dedicated to potential bone replacement material featuring open, micro and interconnected porosity. However, the main challenges of this foaming method is to achieve a homogeneous pore morphology. In a current project, the authors throw light on the bubble/pore and strut formation of this process by in situ computed tomography. This allows for evaluating varying process parameter’s effects on the growth of the ceramic foam during the foaming process. As first result and basis for CT analysis, a stable and reproducible model suspension was developed which resulted in reproducible foam structures. In dependence of selected process parameters like pressure reduction rate or air content in the ceramic suspension resulting Freeze Foams became adjustable with regard to their pore morphology. Pore size and distribution data as well as the porosity were characterized and evaluated accordingly.     

Immunostimulatory CpG oligonucleotides form defined three-dimensional structures: results from an NMR study

The DNA eicosamer 5'-TCCATGACGTTCCTGATGCT-3' is known to stimulate the innate immune system of vertebrae. The immunostimulatory activity is based on the activation of Toll-like receptor 9 (TLR9). While it is known that the CG dinucleotide of the eicosamer has to be unmethylated, the structural basis of the recognition of the DNA through the receptor remains unclear. Oligodeoxynucleotides containing the sequence of the eicosamer, or a portion thereof, ranging in length from hexamer to pentaeicosamer were studied by (1)H NMR spectroscopy. Based on two-dimensional NMR spectra, a number of resonances could be unambiguously assigned. For all oligonucleotides, structural transitions were detected upon heating, as monitored by the line width and chemical shift of low-field resonances. This includes the TC dinucleotide of the 5'-terminal portion, which does not have any clear base-pairing partners. The melting transitions, together with the NOESY cross-peaks, demonstrate that structure formation occurs well beyond the core hexamer 5'-GACGTT-3', a fact that may be important for understanding the molecular recognition by the Toll-like receptors of the innate immune system.     

ProfiSEE Visuelle interaktive Simulation und Modellierung mit dem objektorientierten Programmiersystem Smalltalk-80H

Zusammenfassung Ein wesentliches Problem beim Einsatz von Simulationstechniken ist die Korrektheit des Modells. Mit Hilfe von Visualisierungstechniken wird ein komplexes Simulationsmodell aus dem Bereich der chemischen Produktion dargestellt. Dieses Modell kann interaktiv gesteuert und verändert werden. Simulation und Visualisierung lassen sich mit objektorientierten Systemen auf sehr natürliche Weise beschreiben.Implementiert wurde die Simulation und die Visualisierung mit Hilfe des Smalltalk-80-Systems und des in Smalltalk-80 geschriebenen Hypermedia-Systems The Analyst. Eingesetzt wird es im Bereich Operations Research bei der Ciba-Geigy AG, Basel.

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Summary An essential problem in implementing simulation techniques is the accuracy of the model. With the help of visualization techniques, a complex simulation model from the area of chemical production can be represented.This model can be controlled and altered interactively.Simulation and visualization can be described within object-oriented systems in a very natural way.Simulation and visualization have been implemented with the help of the Smalltalk-80-system and the Hypermedia-System with the name The Analyst written in Smalltalk-80. This is used in the field of operations research at the Ciba-Geigy AG, Basel.