Aryl Hydrocarbon Receptor Activation by Benzo[a]pyrene Prevents Development of Septic Shock and Fatal Outcome in a Mouse Model of Systemic Salmonella enterica Infection.

This study focused on immunomodulatory effects of aryl hydrocarbon receptor (AhR) activation through benzo[a]pyrene (BaP) during systemic bacterial infection. Using a well-established mouse model of systemic Salmonella enterica (S.E.) infection, we studied the influence of BaP on the cellular and humoral immune response and the outcome of disease. BaP exposure significantly reduced mortality, which is mainly caused by septic shock. Surprisingly, the bacterial burden in BaP-exposed surviving mice was significantly higher compared to non-exposed mice. During the early phase of infection (days 1-3 post-infection (p.i.)), the transcription of proinflammatory factors (i.e., IL-12, IFN-γ, TNF-α, IL-1ß, IL-6, IL-18) was induced faster under BaP exposure. Moreover, BaP supported the activity of antigen-presenting cells (i.e., CD64 (FcγRI), MHC II, NO radicals, phagocytosis) at the site of infection. However, early in infection, the anti-inflammatory cytokines IL-10 and IL-22 were also locally and systemically upregulated in BaP-exposed S.E.-infected mice. BaP-exposure resulted in long-term persistence of salmonellae up to day 90 p.i., which was accompanied by significantly elevated S.E.-specific antibody responses (i.e., IgG1, IgG2c). In summary, these data suggest that BaP-induced AhR activation is capable of preventing a fatal outcome of systemic S.E. infection, but may result in long-term bacterial persistence, which, in turn, may support the development of chronic inflammation.

Bayesian modeling of microwave radiometer calibration on the example of the Wendelstein 7-X electron cyclotron emission diagnostic.

This paper reports about a novel approach to the absolute intensity calibration of an electron cyclotron emission (ECE) spectroscopy system. Typically, an ECE radiometer consists of tens of separated frequency channels corresponding to different plasma locations. An absolute calibration of the overall diagnostic including near plasma optics and transmission line is achieved with blackbody sources at LN2 temperature and room temperature via a hot/cold calibration mirror unit. As the thermal emission of the calibration source is typically a few thousand times lower than the receiver noise temperature, coherent averaging over several hours is required to get a sufficient signal to noise ratio. A forward model suitable for any radiometer calibration using the hot/cold method and a periodic switch between them has been developed and used to extract the voltage difference between the hot and cold temperature source via Bayesian analysis. In contrast to the classical analysis which evaluates only the reference temperatures, the forward model takes into account intermediate effective temperatures caused by the finite beam width and thus uses all available data optimally. This allows the evaluation of weak channels where a classical analysis would not be feasible, is statistically rigorous, and provides a measurement of the beam width. By using a variance scaling factor, a model sensitive adaptation of the absolute uncertainties can be implemented, which will be used for the combined diagnostic Bayesian modeling analysis.

Key results from the first plasma operation phase and outlook for future performance in Wendelstein 7-X.

The first physics operation phase on the stellarator experiment Wendelstein 7-X was successfully completed in March 2016 after about 10 weeks of operation. Experiments in this phase were conducted with five graphite limiters as the primary plasma-facing components. Overall, the results were beyond the expectations published shortly before the start of operation [Sunn Pedersen et al., Nucl. Fusion 55, 126001 (2015)] both with respect to parameters reached and with respect to physics themes addressed. We report here on some of the most important plasma experiments that were conducted. The importance of electric fields on global confinement will be discussed, and the obtained results will be compared and contrasted with results from other devices, quantified in terms of the fusion triple product. Expected values for the triple product in future operation phases will also be described and put into a broader fusion perspective.

Preclinical good laboratory practice-compliant safety study to evaluate biodistribution and tumorigenicity of a cartilage advanced therapy medicinal product (ATMP).

BACKGROUND: The clinical development of advanced therapy medicinal products (ATMPs), a new class of drugs, requires initial safety studies that deviate from standard non-clinical safety protocols. The study provides a strategy to address the safety aspects of biodistribution and tumorigenicity of ATMPs under good laboratory practice (GLP) conditions avoiding cell product manipulation. Moreover, the strategy was applied on a human ATMP for cartilage repair. METHODS: The testing strategy addresses biodistribution and tumorigenicity using a multi-step analysis without any cell manipulation to exclude changes of test item characteristics. As a safeguard measurement for meeting regulatory expectations, the project design and goals were discussed continuously with the regulatory authority using a staggered scientific advice concept. Subsequently, the strategy was applied to co.don chondrosphere® (huChon spheroid), a tissue-engineered matrix-free ATMP of human normal chondrocytes. In both the biodistribution and tumorigenicity studies, huChon spheroids were implanted subcutaneously into 40 immunodeficient mice. Biodistribution was studied 1 month after implantation. A skin disc containing the huChon spheroid, two surrounding skin rings and selected organs were analyzed by validated, gender-specific, highly-sensitive triplex qPCR and by immunohistochemistry (IHC). RESULTS: No human DNA was detected in distant skin rings and analyzed organs. IHC revealed no direct or indirect indications of cell migration. Tumorigenicity was assessed 6 months after huChon spheroid implantation by palpation, macroscopic inspection, histology and IHC. No mice from the huChon spheroid group developed a tumor at the implantation site. In two mice, benign tumors were detected that were negative for HLA-ABC, suggesting that they were of spontaneous murine origin. CONCLUSIONS: In summary, the presented strategy using a multi-step analysis was confirmed to be suitable for safety studies of ATMPs.

Characterization of the murine myeloid precursor cell line MuMac-E8.

Starting point for the present work was the assumption that the cell line MuMac-E8 represents a murine cell population with stem cell properties. Preliminary studies already pointed to the expression of stem-cell associated markers and a self-regenerative potential of the cells. The cell line MuMac-E8 should be examined for their differential stage within stem cell hierarchy. MuMac-E8 cells were derived from a chimeric mouse model of arthritis. It could be shown that MuMac-E8 cells express mRNA of some genes associated with pluripotent stem cells (Nanog, Nucleostemin), of genes for hematopoietic markers (EPCR, Sca-1, CD11b, CD45), for the mesenchymal marker CD105 and of genes for the neural markers Pax-6 and Ezrin. In methylcellulose and May-Grünwald-Giemsa staining, hematopoietic colonies were obtained but the hematopoietic system of lethally irradiated mice could not be rescued. Osteogenic differentiation was not detectable. Thus, it became evident that MuMac-E8 represents not a stem cell line. However, MuMac-E8 cells expressed several myeloid surface markers (i.e. CD11b, F4/80, CD14, CD64), showed phagocytosis and is capable of producing nitric oxide. Thus, this cell line seems to be arrested an advanced stage of myeloid differentiation. Adherence data measured by impedance-based real-time cell analysis together with cell morphology data suggested that MuMac-E8 represents a new macrophage precursor cell line exhibiting weak adherence. This cell line is suitable as an in-vitro model for testing of macrophage functions. Moreover, it might be also useful for differentiation or reprogramming studies.

Increase of CD25 expression on bovine neutrophils correlates with disease severity in post-partum and early lactating dairy cows.

Polymorph-nuclear neutrophils (PMN) in cattle exhibit unique features when compared to human or murine PMN and are of particular interest concerning the risk of post-partum mammary gland or extra-mammary infections related to the periparturient suppression of neutrophil functions. Former studies could show that effects of IL-2 on innate immune cells such as PMN were mediated by the interleukin-2 receptor (IL-2R) ß and γ chains. In the current study we could detect IL-2Rα (CD25) expression on bovine PMN using flow-cytometric analysis. CD25 was detected on granulocytes from post-partum and early lactating cows with different inflammatory conditions. The expression of CD25 on PMN in blood and raw milk increased with disease severity. Our results suggest CD25 expression on PMN as a potential biomarker for acute infections in cattle. Furthermore, our data provide a basis to better understanding of the periparturient functional suppressions of PMN that might reveal new molecular targets for therapy or prevention of disease.

Identification and evaluation of novel synovial tissue biomarkers in rheumatoid arthritis by laser scanning cytometry.

INTRODUCTION: Suitable biomarkers are essential for therapeutic strategies in personalized medicine in terms of diagnosis as well as of prognosis. With highly specific biomarkers, it is possible, for example, to identify patients with poor prognosis, which enables early intervention and intensive treatment. The aim of this study was to identify and validate biomarkers and possible combinations for a prospective use in immunoscintigraphy, which may improve diagnosis of rheumatoid arthritis (RA) patients with consideration of inflammatory activity in the affected joints. Therefore, we tested several monoclonal antibodies (mAbs) directed against cellular-surface molecules on cells likely to be involved in the pathogenesis of RA. METHODS: Synovial tissue from patients with long-standing RA (accompanied by synovitis with varying states of current activity) and patients with acute non-RA arthritis were stained for surface molecules on different cell types by using fluorochrome-labeled antibodies. Tissue analysis was done by laser scanning cytometry (LSC), and statistical evaluation, by discriminant analysis and ROC analysis. RESULTS: CD11b, HLA-DR, CD90, and CD64 revealed significant differences between tissues from patients with RA and acute non-RA arthritis. Especially with the expression of CD64, both patient cohorts could be discriminated with high sensitivity and specificity. RA classification was improved by simultaneously investigating the expression of two or three different surface proteins, such as HLA-DR, CD90, and CD29 in the tissue. The simultaneous analysis of CD64 together with CD304 or the combination of CD11b and CD38 was suitable for the identification of RA patients with high current activity in synovitis. CONCLUSIONS: In this study, we showed that LSC is a novel reliable method in biomarker prevalidation in RA. Hence, identified mAbs in situ may allow their potential use in in vivo approaches. Moreover, we proved that biomarker-combination analysis resulted in better discrimination than did single-marker analysis. Combinations of these markers make a novel and reliable panel for the discrimination between RA and acute non-RA arthritis. In addition, further expedient combinations may be novel promising biomarker panels to identify current activity in synovitis in RA.

Evaluation of the preventive capacities of a topically applied azithromycin formulation against Lyme borreliosis in a murine model.

OBJECTIVES: Systemic antibiotic treatment of Lyme borreliosis is effective during the early stages of the infection, while chronic manifestations of the disease may remain refractory and difficult to treat. This study was carried out in order to evaluate the potential of topically applied azithromycin to eliminate the spirochaetal organisms in the skin of the freshly bitten host and thereby prevent Lyme borreliosis. METHODS: Laboratory mice were challenged with Borrelia burgdorferi sensu stricto by needle inoculation or via infected ticks as vectors. Then, an azithromycin-containing formulation was applied once daily to the sites of exposure for three consecutive days. In the case of needle inoculation, a 5% azithromycin formulation was applied starting 1 h, 3 days and 5 days after infection. In the case of tick exposure, 4%, 10% and 20% azithromycin formulations were applied, starting directly after the detachment of the engorged ticks. Subsequently, the infection status of the mice was determined. RESULTS: Concentrations of azithromycin in murine skin were >3800-fold higher than the published minimal inhibitory concentration for B. burgdorferi as soon as 3 h after the first application. After needle inoculation, spirochaetes were not detectable in all infected mice after treatment, if the first application started 1 h or even after 3 days post-infection. Furthermore, no borrelial organisms were detected after topical treatment when ticks were used for spirochaete inoculation. CONCLUSIONS: Our data indicate that topical treatment with a formulation containing azithromycin is a promising approach to prevent Lyme borreliosis shortly after a tick bite.

Metamorphosis of Borrelia burgdorferi organisms--RNA, lipid and protein composition in context with the spirochetes' shape.

Borrelia burgdorferi, the agent of Lyme borreliosis, has the ability to undergo morphological transformation from a motile spirochetal to non-motile spherical shape when it encounters unfavorable conditions. However, little information is available on the mechanism that enables the bacterium to change its shape and whether major components of the cells--nucleic acids, proteins, lipids--are possibly modified during the process. Deducing from investigations utilizing electron microscopy, it seems that shape alteration begins with membrane budding followed by folding of the protoplasmatic cylinder inside the outer surface membrane. Scanning electron microscopy confirmed that a deficiency in producing functioning periplasmic flagella did not hinder sphere formation. Further, it was shown that the spirochetes' and spheres' lipid compositions were indistinguishable. Neither phosphatidylcholine nor phosphatidylglycerol were altered by the structural transformation. In addition, no changes in differential protein expression were detected during this process. However, minimal degradation of RNA and a reduced antigen-antibody binding activity were observed with advanced age of the spheres. The results of our comparisons and the failure to generate mutants lacking the ability to convert to spheres suggest that the metamorphosis of B. burgdorferi results in a conditional reconstruction of the outer membrane. The spheres, which appear to be more resistant to unfavorable conditions and exhibit reduced immune reactivity when compared to spirochetes, might allow the B. burgdorferi to escape complete clearance and possibly ensure long-term survival in the host.

Borrelia burgdorferi sensu lato species in Europe induce diverse immune responses against C6 peptides in infected mice.

The diversity of Lyme-borreliosis-inducing Borrelia species in Europe set high standards for the use of serodiagnostic test systems in terms of specificity and sensitivity. In the United States, the one-step C6 antibody test system based on the invariable domain IR6 of the VlsE molecule has been established as a successful diagnostic tool for testing canine samples. However, only a limited set of data are available regarding the antigenicity of the C6 peptides in an experimental murine model and sensitivity of the test regarding European Borrelia species. In order to investigate antibody reactions induced by these spirochetes, a total of 142 C3H/HeN mice were inoculated with Borrelia burgdorferi sensu stricto N40, B. garinii PBi, two isolates of B. afzelii, B. spielmanii A14S, B. valaisiana Rio6, B. valaisiana VS116, or B. lusitaniae. Infection of the mice was documented utilizing tissue culture and PCR. The IR6 sequences of B. burgdorferi sensu stricto B31, B. garinii IP90, and two B. afzelii ACAI strains have been used to synthesize and test additional C6 peptides. Compared to the well-established two-tiered test system, the results indicate that single C6 peptides derived from B. burgdorferi sensu stricto and B. garinii can be used in an enzyme-linked immunosorbent assay-based technique to detect murine antibodies induced by either agent. Little is known about the prevalence or pathogenicity of the B. afzelii strains in mammalian hosts, but our experimental data indicate differences in the C6 peptide test sensitivity for the detection of antibodies induced by different strains or isolates of B. afzelii.

Protective immunity to systemic infection with attenuated Salmonella enterica serovar enteritidis in the absence of IL-12 is associated with IL-23-dependent IL-22, but not IL-17.

IL-12 is essential for protective T cell-mediated immunity against Salmonella infection. To characterize the role of the related cytokine IL-23, wild-type (WT) C57BL/6 and p19(-/-) mice were infected systemically with an attenuated strain of Salmonella enterica serovar Enteritidis (S. Enteritidis). IL-23-deficient mice controlled infection with S. Enteritidis similarly as WT mice. Similar IFN-gamma production as compared with WT mice, but defective IL-17A and IL-22 production was found in the absence of IL-23. Nevertheless, although IL-23 is required for T cell-dependent cytokine responses, IL-23 is dispensable for protection against S. Enteritidis when IL-12 is present. To analyze the role of IL-23 in the absence of IL-12, low doses of S. Enteritidis were administered to p35(-/-) mice (lacking IL-12), p35/19(-/-) mice (lacking IL-12 and IL-23), p35/40(-/-) mice (lacking IL-12, IL-23, and homodimeric IL-12p40), or p35/IL-17A(-/-) mice (lacking IL-12 and IL-17A). We found survival of p35(-/-) and p35/IL-17A(-/-) mice, whereas p35/19(-/-) and p35/40(-/-) mice died within 3-6 wk and developed liver necrosis. This indicates that IL-23, but not homodimeric IL-12p40, is required for protection, which, surprisingly, is independent of IL-17A. Moreover, protection was associated with IL-22, but not IL-17F or IL-21 expression or with neutrophil recruitment. Finally, anti-IL-22 treatment of S. Enteritidis-infected p35(-/-) mice resulted in liver necrosis, indicating a central role of IL-22 in hepatocyte protection during salmonellosis. In conclusion, IL-23-dependent IL-22, but not IL-17 production is associated with protection against systemic infection with S. Enteritidis in the absence of IL-12.

Borrelia burgdorferi potently activates bone marrow-derived conventional dendritic cells for production of IL-23 required for IL-17 release by T cells.

Lyme borreliosis is characterized by cellular inflammatory responses at multiple body sites. Recently, an association of interleukin-17 (IL-17) and Lyme arthritis was suggested. In this context, it is of special interest that the heterodimeric cytokine IL-23 can act on T cells and initiate the up-regulation of effector cytokines such as IL-17. To determine the role of this specific cytokine cascade for the induction of subsequently induced proinflammatory events we developed an in vitro system to investigate the IL-23-inducing capacity of Borrelia burgdorferi and the potential of the spirochete for inducing the IL-23/IL-17 axis. We used cells derived from mice deficient for IL-23 or IL-12 only or deficient for both IL-12 and IL-23 to define precisely the function of these cytokines. Experiments with bone marrow-derived dendritic cells (BMDC) identified these cells as sources for IL-23 but not for IL-12 after B. burgdorferi exposure. Subsequent investigations with T cell-depleted splenocyte fractions revealed a tight IL-23/IL-17 axis in response to the spirochetes. Monoclonal antibodies that block IL-23 showed further that BMDC-derived IL-23 was required for production of IL-17 in this experimental model. These in vitro data describing a spirochete-induced release of IL-23 may help to define IL-17-dependent inflammatory responses in the disease.

Borrelia burgdorferi organisms lacking plasmids 25 and 28-1 are internalized by human blood phagocytes at a rate identical to that of the wild-type strain.

Lyme borreliosis caused by Borrelia burgdorferi is a persistent infection capable of withstanding the host's vigorous immune response. Several reports have shown that the spirochete's linear plasmids 25 and 28-1 are essential for its infectivity. In this context, it was proposed that Borrelia burgdorferi organisms control their uptake by macrophages and polymorphonuclear leukocytes (PMNs) through plasmid-encoded proteins and that this mechanism confers resistance to phagocytosis. To investigate this proposal, a precise flow-cytometry-based method with human blood was used to study the impact of the plasmids 25 and 28-1 on B. burgdorferi clearance over 150 min and to investigate whether low-passage organisms are more resistant to phagocytosis than high-passage B. burgdorferi. Exposure of human blood PMNs or blood monocytes to fluorescein isothiocyanate-labeled B. burgdorferi B31 organisms lacking the linear plasmids 25, 28-1, or both revealed that all spirochete populations were internalized at the same rate as the wild-type borrelia parent strain B31. Moreover, no differences in phagocytosis kinetics were detected when low- or high-passage wild-type B. burgdorferi B31 or N40 were cocultured with blood cells. Plasmid loss and probable associated surface protein changes due to serial in vitro propagation of B. burgdorferi do not affect the resistance of these organisms to internalization by phagocytic cells. In particular, we found no evidence for a plasmid-controlled (lp25 and lp28-1) resistance of B. burgdorferi to phagocytosis by leukocytes of the host's innate immune system.

Calcitonin receptor-like receptor is expressed on gastrointestinal immune cells.

BACKGROUND/AIMS: Pharmacological and morphological studies suggest that the gut mucosal immune system and local neuropeptide-containing neurones interact. We aimed to determine whether gut immune cells are targets for calcitonin gene-related peptide (CGRP), which has potent immune regulatory properties. METHODS: Using density gradient centrifugation, rat lamina propria mononuclear cells (LP-MNCs) and intra-epithelial lymphocytes (IELs) were isolated. RT-PCR was employed for the detection of mRNA of rat calcitonin receptor-like receptor (CRLR), which is considered to represent the pharmacologically defined CGRP receptor-1 subtype, as well as mRNA of the receptor activity-modifying proteins, which are essential for CRLR function and determine ligand specificity. A radioreceptor assay was employed for the detection of specific CGRP binding sites. RESULTS: RT-PCR and DNA sequencing showed that LP-MNCs and IELs express CRLR. Incubation of isolated LP-MNCs with radiolabelled alphaCGRP revealed the existence of specific binding sites for CGRP. CONCLUSION: These novel data indicate that mucosal immune cells of the rat gut are a target for CGRP and provide significant evidence that CGRP functions as an immune regulator in the gut mucosa.