Mycoplasma pneumoniae Compared to Streptococcus pneumoniae Avoids Induction of Proinflammatory Epithelial Cell Responses despite Robustly Inducing TLR2 Signaling.

Mycoplasma pneumoniae and Streptococcus pneumoniae are the most common bacterial causes of pneumonia in children. The clinical characteristics of pneumonia differ significantly between the two bacteria. We aimed to elucidate the differences in pathogenesis between M. pneumoniae and S. pneumoniae by characterizing the respiratory epithelial cell immune response to both pathogens. Using primary human bronchial epithelial cells in air-liquid interface cultures, we observed lower production of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 in response to M. pneumoniae than to S. pneumoniae. In contrast to the differences in proinflammatory cytokine production, Toll-like receptor 2 (TLR2)-mediated signaling in response to M. pneumoniae was stronger than to S. pneumoniae. This difference largely depended on TLR1 and not TLR6. We found that M. pneumoniae, but not S. pneumoniae, also induced signaling of TLR10, a coreceptor of TLR2 that has inhibitory properties. M. pneumoniae-induced TLR10 signaling on airway epithelial cells was partially responsible for low IL-8 production, as blocking TLR10 by specific antibodies increased cytokine production. M. pneumoniae maintained Th2-associated cytokine production by epithelial cells, which concurs with the known association of M. pneumoniae infection with asthma. M. pneumoniae left IL-33 levels unchanged, whereas S. pneumoniae downregulated IL-33 production both under homeostatic and Th2-promoting conditions. By directly comparing M. pneumoniae and S. pneumoniae, we demonstrate that M. pneumoniae avoids induction of proinflammatory cytokine response despite its ability to induce robust TLR2 signaling. Our new findings suggest that this apparent paradox may be partially explained by M. pneumoniae-induced signaling of TLR2/TLR10.

Quantification of silane molecules on oxidized silicon: are there options for a traceable and absolute determination?

Organosilanes are used routinely to functionalize various support materials for further modifications. Nevertheless, reliable quantitative information about surface functional group densities after layer formation is rarely available. Here, we present the analysis of thin organic nanolayers made from nitrogen containing silane molecules on naturally oxidized silicon wafers with reference-free total reflection X-ray fluorescence (TXRF) and X-ray photoelectron spectroscopy (XPS). An areic density of 2-4 silane molecules per nm(2) was calculated from the layer's nitrogen mass deposition per area unit obtained by reference-free TXRF. Complementary energy and angle-resolved XPS (ER/AR-XPS) in the Si 2p core-level region was used to analyze the outermost surface region of the organic (silane layer)-inorganic (silicon wafer) interface. Different coexisting silicon species as silicon, native silicon oxide, and silane were identified and quantified. As a result of the presented proof-of-concept, absolute and traceable values for the areic density of silanes containing nitrogen as intrinsic marker are obtained by calibration of the XPS methods with reference-free TXRF. Furthermore, ER/AR-XPS is shown to facilitate the determination of areic densities in (mono)layers made from silanes having no heteroatomic marker other than silicon. After calibration with reference-free TXRF, these areic densities of silane molecules can be determined when using the XPS component intensity of the silane's silicon atom.

Lateral resolution of nanoscaled images delivered by surface-analytical instruments: application of the BAM-L200 certified reference material and related ISO standards.

The certified reference material BAM-L200, a nanoscale stripe pattern for length calibration and specification of lateral resolution, is described. BAM-L200 is prepared from a cross-sectioned epitaxially grown layer stack of AlxGa1-xAs and InxGa1-xAs on a GaAs substrate. The surface of BAM-L200 provides a flat pattern with stripe widths ranging down to 1 nm. Calibration distances, grating periods and stripe widths have been certified by TEM with traceability to the length unit. The combination of gratings, isolated narrow stripes and sharp edges of wide stripes offers plenty of options for the determination of lateral resolution, sharpness and calibration of length scale at selected settings of imaging surface-analytical instruments. The feasibility of the reference material for an analysis of the lateral resolution is demonstrated in detail by evaluation of ToF-SIMS, AES and EDX images. Other applications developed in the community are summarized, too. BAM-L200 fully supports the implementation of the revised International Standard ISO 18516 (in preparation) which is based on knowledge outlined in the Technical Report ISO/TR 19319:2013.

Lattice QCD phase diagram in and away from the strong coupling limit.

We study lattice QCD with four flavors of staggered quarks. In the limit of infinite gauge coupling, "dual" variables can be introduced, which render the finite-density sign problem mild and allow a full determination of the µ-T phase diagram by Monte Carlo simulations, also in the chiral limit. However, the continuum limit coincides with the weak coupling limit. We propose a strong-coupling expansion approach towards the continuum limit. We show first results, including the phase diagram and its chiral critical point, from this expansion truncated at next-to-leading order.

Comprehensive comparison of various techniques for the analysis of elemental distributions in thin films.

The present work shows results on elemental distribution analyses in Cu(In,Ga)Se2 thin films for solar cells performed by use of wavelength-dispersive and energy-dispersive X-ray spectrometry (EDX) in a scanning electron microscope, EDX in a transmission electron microscope, X-ray photoelectron, angle-dependent soft X-ray emission, secondary ion-mass (SIMS), time-of-flight SIMS, sputtered neutral mass, glow-discharge optical emission and glow-discharge mass, Auger electron, and Rutherford backscattering spectrometry, by use of scanning Auger electron microscopy, Raman depth profiling, and Raman mapping, as well as by use of elastic recoil detection analysis, grazing-incidence X-ray and electron backscatter diffraction, and grazing-incidence X-ray fluorescence analysis. The Cu(In,Ga)Se2 thin films used for the present comparison were produced during the same identical deposition run and exhibit thicknesses of about 2 µm. The analysis techniques were compared with respect to their spatial and depth resolutions, measuring speeds, availabilities, and detection limits.

Accumulation of autoreactive effector T cells and allo-specific regulatory T cells in the pancreas allograft of a type 1 diabetic recipient.

AIMS/HYPOTHESIS: Simultaneous kidney-pancreas transplantation is an established treatment for patients with type 1 diabetes and end-stage renal failure, even though restored beta cell function may become affected by recurrent islet autoimmunity or graft rejection. We characterised infiltrating lymphocytes isolated from a pancreatic graft with normal endocrine function in a kidney-pancreas recipient with type 1 diabetes. METHODS: The pancreas graft was removed due to recurrent graft pancreatitis of unknown cause. Pancreas-infiltrating lymphocytes and peripheral blood mononuclear cells (PBMC) were isolated and characterised phenotypically and functionally. RESULTS: Compared with PBMC, pancreas-infiltrating lymphocytes exhibited a distinct activation/memory phenotype and T cell receptor profile that were indicative of selective infiltration of the pancreas. Islet autoreactive CD8(+) T cells could be detected in the pancreas and were increased in frequency compared with PBMC. Additionally, an augmentation of CD8(+) CD28(-) regulatory T cells was observed in the pancreas; these induced expression of the inhibitory receptor immunoglobulin-like transcript-3 on antigen-presenting cells in a donor HLA class I-specific manner. CONCLUSIONS/INTERPRETATION: These data demonstrate the simultaneous presence of regulatory and effector T cells in the pancreas allograft of a recipient with type 1 diabetes. They also indicate that circulating islet autoreactive T cells may reflect immunological processes in pancreatic tissue, even though their frequency in the periphery may lead to underestimation of their presence in the pancreas. Additional specificities were also present in the pancreas that were undetectable in the circulation.

Differential inhibition of autoreactive memory- and alloreactive naive T cell responses by soluble cytotoxic T lymphocyte antigen 4 (sCTLA4), CTLA4Ig and LEA29Y.

Cytotoxic T lymphocyte antigen 4 (CTLA4) is a potent inhibitory co-stimulatory molecule believed to be involved in type 1 diabetes and other autoimmune diseases. An association has been reported of both mRNA expression and serum levels of the soluble splice variant of CTLA4 (sCTLA4) with type 1 diabetes. Furthermore, recombinant fusion proteins CTLA4Ig and LEA29Y have been proposed as therapies for type 1 diabetes. We studied the role of (s)CTLA4 in islet autoimmunity. Binding capacity of the proteins to antigen-presenting cells was determined by flow cytometry in competition and binding assays. Functionality of sCTLA4 as well as the therapeutic inhibitory fusion proteins CTLA4Ig and LEA29Y was measured in a dose-response lymphocyte stimulation test, using a panel of diabetes-associated T cell clones reactive to islet autoantigens. As controls, mixed lymphocyte reactions (MLR) were performed to assess functionality of these proteins in a primary alloreactive setting. All three CTLA4 molecules were able to bind to antigen-presenting cells and inhibit the expression of CD80/CD86. sCTLA4 was able to suppress proliferation of different committed autoreactive T cell clones in a dose-dependent manner, whereas CTLA4Ig and LEA29Y were not. Conversely, CTLA4Ig and LEA29Y, rather than sCTLA4, were able to suppress naive alloreactive proliferation in a MLR. Our results indicate a differential role for sCTLA4, CTLA4Ig and LEA29Y proteins in memory versus primary immune responses with implications for efficacy in intervention therapy.

Human clonal CD8 autoreactivity to an IGRP islet epitope shared between mice and men.

Type 1 diabetes (T1D) is a multifactorial disease characterized by the infiltration and subsequent destruction of the pancreatic insulin-producing beta cells by autoreactive T cells. CD8(+) T cells play an essential role in this beta cell destruction. However, little is known about the target antigens of CD8(+) T cells in human T1D patients. The aim of this study was to assess whether an epitope derived from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), IGRP(265-273,) which has recently been identified as a target in non-obese diabetic (NOD) mice and is fully homologous to the human epitope, is a target of human diabetogenic CD8(+) T cells. We isolated a human CD8 T cell clone against this epitope, which confirms that this IGRP epitope is shared across species.

A sealable ultrathin window sample cell for the study of liquids by means of soft X-ray spectroscopy.

A new sample cell concept for the analysis of liquids or solid-liquid interfaces using soft X-ray spectroscopy is presented, which enables the complete sealing of the cell as well as the transport into vacuum via, for example, a load-lock system. The cell uses pressure monitoring and active as well as passive pressure regulation systems, thereby facilitating the full control over the pressure during filling, sealing, evacuation, and measurement. The cell design and sample preparation as well as the crucial sealing procedure are explained in detail. As a first proof-of-principle experiment, successful nitrogen K-edge fluorescence yield near-edge X-ray absorption fine structure experiments of a biomolecular solution are presented. For this purpose, it is shown that the careful evaluation of all involved parameters, such as window type or photon flux, is desirable for optimizing the experimental result.

Real Time Imaging of Deuterium in a Duplex Stainless Steel Microstructure by Time-of-Flight SIMS.

For more than one century, hydrogen assisted degradation of metallic microstructures has been identified as origin for severe technical component failures but the mechanisms behind have not yet been completely understood so far. Any in-situ observation of hydrogen transport phenomena in microstructures will provide more details for further elucidation of these degradation mechanisms. A novel experiment is presented which is designed to elucidate the permeation behaviour of deuterium in a microstructure of duplex stainless steel (DSS). A hydrogen permeation cell within a TOF-SIMS instrument enables electrochemical charging with deuterium through the inner surface of the cell made from DSS. The outer surface of the DSS permeation cell exposed to the vacuum has been imaged by TOF-SIMS vs. increasing time of charging with subsequent chemometric treatment of image data. This in-situ experiment showed evidently that deuterium is permeating much faster through the ferrite phase than through the austenite phase. Moreover, a direct proof for deuterium enrichment at the austenite-ferrite interface has been found.

The effect of finasteride, a 5 alpha-reductase inhibitor, on scalp skin testosterone and dihydrotestosterone concentrations in patients with male pattern baldness.

The effects of the 5 alpha-reductase inhibitor, finasteride, on scalp skin testosterone (T) and dihydrotestosterone (DHT) levels were studied in patients with male pattern baldness. In a double blind study, male patients undergoing hair transplantation were treated with oral finasteride (5 mg/day) or placebo for 28 days. Scalp skin biopsies were obtained before and after treatment for measurement of T and DHT by high pressure liquid chromatography-RIA. In 10 male subjects studied at baseline, mean (+/- SEM) DHT levels were significantly higher in bald (7.37 +/- 1.24 pmol/g) compared to hair-containing (4.20 +/- 0.65 pmol/g) scalp, whereas there was no difference in mean T levels at baseline. In bald scalp from 8 patients treated with finasteride, the mean DHT concentration decreased from 6.40 +/- 1.07 pmol/g at baseline to 3.62 +/- 0.38 pmol/g on day 28. Scalp T levels increased in 6 of 8 subjects treated with finasteride. Finasteride decreased the mean serum DHT concentration from 1.36 +/- 0.18 nmol/L (n = 8) at baseline to 0.46 +/- 0.10 nmol/L on day 28 and had no effect on serum T. There were no significant changes in scalp or serum T or DHT in placebo-treated patients. In this study, male subjects treated with 5 mg/day finasteride for 4 weeks had significantly decreased concentrations of DHT in bald scalp, resulting in a mean level similar to the baseline levels found in hair-containing scalp.

Distribution and origin of calcitonin gene-related peptide (CGRP) immunoreactivity in the sensory innervation of the mammalian eye.

The occurrence, distribution, and origin of immunoreactive calcitonin gene-related peptide (CGRP) in nerves of rat, guinea pig, cat, and monkey eyes were investigated by immunocytochemistry, radioimmunoassay, and chromatography. A rich network of CGRP-immunoreactive nerve fibres was noted in the anterior uvea, which was widely distributed in both dilator and constrictor pupillae muscles and extended to the ciliary body and uveal blood vessels. Numerous CGRP-immunoreactive neuronal cells were present in the trigeminal ganglion. The extractable CGRP was 8.6 +/- 1.8 pmoles/gm of tissue in the iris and 44.0 +/- 8.1 pmoles/gm in the trigeminal ganglion. Following damage to the Gasserian ganglion a marked decrease of CGRP immunoreactivity was observed in the anterior uvea (control 11.3 +/- 1.6 pmoles/gm; operated 1.4 +/- 0.1 pmoles/gm) confirming the origin of the immunoreactive fibres from trigeminal primary sensory neurons. The sensory nature of the CGRP-immunoreactive fibres was substantiated by the depletion of CGRP immunoreactivity observed after treatment with capsaicin, which is known to cause selective degeneration of sensory neurons. Comparative studies on the distribution and colocalisation of CGRP and the putative sensory neurotransmitter substance P revealed a closely parallel distribution of the two peptides in certain regions of the uvea and their coexistence in a subpopulation of trigeminal primary sensory neurons. This study suggests that the sensory nervous system in the eye is more heterogeneous in terms of its putative neurotransmitters than previously indicated.

Regional distribution of lysosomal enzymes in the canine retinal pigment epithelium.

We have investigated the activities of four lysosomal enzymes in RPE cells isolated from three regions of the canine fundus: the tapetal area, the central pigmented area and the peripheral area. The results obtained with freshly isolated cells showed that the activities of acid phosphatase, B-glucuronidase and N-acetyl-B-glucosaminidase were significantly higher in RPE cells derived from the peripheral region when compared to those from the two central regions. In contrast, the activity of cathepsin D was significantly higher in the tapetal region than in the periphery. The regional distribution of both acid phosphatase and B-glucuronidase observed in fresh RPE cells was progressively lost when these cells were grown in culture. Estimations of photoreceptor density per RPE cell from each of the regions indicated that the number of photoreceptors per RPE cell did not vary significantly with retinal location and suggested that variations in enzyme content were not related to differences in photoreceptor cell distribution.

Measuring changes in logarithmic data, with special reference to bronchial responsiveness.

Bronchial provocation tests with agents such as histamine and methacholine are commonly used in clinical and epidemiological studies of respiratory illness because bronchial hyperresponsiveness is a non-specific abnormality of the airways which is characteristic of asthma. However, measurements of bronchial responsiveness are log-normally distributed. As a result, special considerations need to be given to reporting within-subject changes in these measurements in longitudinal studies as, for example, in clinical trials or in any study in which a before-and-after experimental design is used. In these types of experiments, changes in bronchial responsiveness should not be simply expressed in the units of the measurement, such as dose of provoking agent, but must be expressed in units based on a logarithmic scale. The appropriate log-based units for measuring within-subject changes are doubling dose, fold difference or percent change. This paper explains the methods for calculating changes in these units in a statistically correct manner. All three units represent different ways of expressing the same change on a logarithmic scale. However, 'doubling dose' is only appropriate when it relates directly to the method of administering the provoking agent in doubling concentrations and 'fold difference' or 'percent change' are both appropriate for expressing any log-based changes. The methods for calculating changes in these units also apply to calculations of repeatability within test methods and to calculations of comparability and agreement between test methods. The methods are described solely for reporting changes in units of bronchial responsiveness but are applicable to other log-normally distributed measurements.

Presence and actions of vasopressin-like peptides in the rabbit anterior uvea.

The presence of vasopressin-like immunoreactivity (VP-IR) in the rabbit eye was demonstrated by radioimmunoassay. Trigeminal nerve denervation resulted in a significant and selective decrease in the levels of VP-IR in the iris sphincter muscle and the cornea. The isolated iris sphincter muscle contracted in response to low concentrations of [Arg8]vasopressin (AVP) and related peptides. The V1 vasopressin receptor antagonist, d(CH2)5Tyr(Me)AVP, potently inhibited the contractile responses to AVP. AVP was found to induce an increase in the accumulation of inositol phosphates in the iris sphincter muscle but not in the dilator/ciliary body preparation in vitro. The present investigation demonstrates the presence of VP-IR in the rabbit eye and that this substance may be another sensory nerve-derived mediator acting on specific target sites in the anterior uvea.

Midline alopecia reduction combined with hair transplantation.

The advantages gained by incorporating alopecia reduction (AR) in patients undergoing hair transplantation, or with patients who have previously had hair transplantation, are delineated. The specific advantages of the midline elliptical pattern are discussed. Clinical considerations that determine whether ARs are necessary are the degree of baldness, laxity of the scalp, age of the patient, and ultimate goal of the individual. The advantages and disadvantages of performing ARs or hair transplantation first are discussed and the new concept of minireductions also outlined.

Corticosteroids in the treatment of alopecia totalis. Systemic effects.

Fifteen patients with alopecia totalis (AT) or alopecia universalis (AU) were treated with combined topical, intralesional, and oral corticosteroids. All or virtually all scalp hair regrown on seven patients, and they have been able to discontinue oral corticosteroids without recurrence of AT or AU for periods of three months to 7 1/2 years, with an average remission of 32 months. Two additional patients are currently receiving 5 mg of prednisone every two days. Using topical and intralesional corticosteroids in more resistant areas allowed for more rapid lowering of oral doses and thus side effects were minimized. In view of the usually nearly hopeless prognosis for AT and AU and the results reported here, a trial course of topical, intralesional, and oral corticosteroid treatment would seem to be reasonable for highly motivated patients.

Neuropeptide Y-immunoreactive nerves in the uvea of guinea pig and rat.

Neuropeptide Y (NPY)-immunoreactivity has been localized to a rich network of fibres in the uvea of guinea pig and rat. In the iris, NPY-immunoreactive nerves were present in iridial smooth muscle and around blood vessels; similarly throughout the choroid a dense network of NPY-immunoreactive fibres was found surrounding the choroidal vascularization. Following sympathectomy a marked decrease of NPY-immunoreactivity, in particular in the iris and less marked in the choroid, was noted, indicating that these fibres may originate at least in part from neuronal cell bodies in the superior cervical ganglion. The close relationship of the NPY-immunoreactive fibres to blood vessels in both anterior and posterior uvea is consistent with the established vasoconstrictor action of this peptide.