Impact of immunosuppressive and antifungal drugs on PBMC- and whole blood-based flow cytometric CD154+ Aspergillus fumigatus specific T-cell quantification.

Flow cytometric quantification of CD154+ mould specific T-cells in antigen-stimulated peripheral blood mononuclear cells (PBMCs) or whole blood has been described as a supportive biomarker to diagnose invasive mould infections and to monitor therapeutic outcomes. As patients at risk frequently receive immunosuppressive and antifungal medication, this study compared the matrix-dependent impact of representative drugs on CD154+ T-cell detection rates. PBMCs and whole blood samples from healthy adults were pre-treated with therapeutic concentrations of liposomal amphotericin B, voriconazole, posaconazole, cyclosporine A (CsA) or prednisolone. Samples were then stimulated with an Aspergillus fumigatus lysate or a viral antigen cocktail (CPI) and assessed for CD154+ T-helper cell frequencies. Specific T-cell detection rates and technical assay properties remained largely unaffected by exposure of both matrices to the studied antifungals. By contrast, CsA and prednisolone pre-treatment of isolated PBMCs and whole blood adversely impacted specific T-cell detection rates and caused elevated inter-replicate variation. Unexpectedly, the whole blood-based protocol that uses additional α-CD49d co-stimulation was less susceptible to CsA and prednisolone despite prolonged drug exposure in the test tube. Accordingly, addition of α-CD49d during PBMC stimulation partially attenuated the impact of immunosuppressive drugs on test performance. Translating these results into the clinical setting, false-negative results of CD154+ antigen-specific T-cell quantification need to be considered in patients receiving T-cell-active immunosuppressive medication. Optimized co-stimulation regimes with α-CD49d could contribute to an improved feasibility of functional T-cell assays in immunocompromised patient populations.

Development and evaluation of a whole blood-based approach for flow cytometric quantification of CD154+ mould-reactive T cells.

CD154+ mould-reactive T cells were proposed as a novel biomarker in the diagnosis of invasive mycoses. As PBMC-based protocols for flow cytometric quantification of these cells are logistically challenging and susceptible to preanalytic delays, this study evaluated and optimized a whole blood-based method for the detection of mould-reactive T cells. Blood collection tubes containing costimulatory antibodies and Aspergillus fumigatus mycelial lysates were inoculated with heparinized whole blood from healthy adults, and detection rates of CD154+/CD4+A. fumigatus reactive T cells were compared with PBMC-based detection using samples from the same donors. In contrast to the PBMC-based method, double costimulation with αCD28 and αCD49d was crucial for reliable whole blood stimulation. Optimizing stimulation schemes for both matrixes, significantly higher specific T-cell detection rates were achieved by the whole blood-based method, whereas the unspecific background stimulation remained low. MHC II-dependent CD154+ upregulation was demonstrated for both matrixes. Excellent correlation and reproducible conversion factors between whole blood and PBMC-based results were observed. Using frozen ready-to-use test tubes containing costimulatory antibodies and lysates, detection rates of specific T cells were comparable to freshly prepared blood collection tubes. The optimized whole blood-based protocol was also used to detect Rhizopus arrhizus and Rhizomucor pusillus reactive T cells, resulting in 1.5- to 2.7-fold higher detection rates compared with PBMC-based measurement. In summary, the whole blood protocol is a robust, highly sensitive, and cost-effective method for mould-reactive T-cell quantification, allowing for point-of-care sample stimulation and contributing to better assay standardization in multi-centre evaluation of mould reactive T-cell quantification.

Effects of Spacers on Photoinduced Reversible Solid-to-Liquid Transitions of Azobenzene-Containing Polymers.

Photoisomerization in some azobenzene-containing polymers (azopolymers) results in reversible solid-to-liquid transitions because trans- and cis-azopolymers have different glass transition temperatures. This property enables photoinduced healing and processing of azopolymers with high spatiotemporal resolution. However, a general lack of knowledge about the influence of the polymer structure on photoinduced reversible solid-to-liquid transitions hinders the design of such novel polymers. Herein, the synthesis and photoresponsive behavior of new azopolymers with different lengths of spacers between the polymer backbone and the azobenzene group on the side chain are reported. Azopolymers with no and 20 methylene spacers did not show photoinduced solid-to-liquid transitions. Azopolymers with 6 or 12 methylene spacers showed photoinduced solid-to-liquid transitions. This study demonstrates that spacers are essential for azopolymers with photoinduced reversible solid-to-liquid transitions, and thus, gives an insight into how to design azopolymers for photoinduced healing and processing.

Evaluation of Aspergillus and Mucorales specific T-cells and peripheral blood mononuclear cell cytokine signatures as biomarkers of environmental mold exposure.

Mold specific T-cells have been described as a supportive biomarker to monitor invasive mycoses and mold exposure. This study comparatively evaluated frequencies and cytokine profiles of Aspergillus fumigatus and Mucorales reactive T-cells depending on environmental mold exposure. Peripheral blood mononuclear cells (PBMCs) obtained from 35 healthy donors were stimulated with mycelial lysates of A. fumigatus and three human pathogenic Mucorales species. CD154+ specific T-cells were quantified by flow cytometry. In a second cohort of 20 additional donors, flow cytometry was complemented by 13-plex cytokine assays. Mold exposure of the subjects was determined using a previously established questionnaire. Highly exposed subjects exhibited significantly greater CD154+A. fumigatus and Mucorales specific naïve and memory T-helper cell frequencies. Significant correlation (r = 0.48 - 0.79) was found between A. fumigatus and Mucorales specific T-cell numbers. Logistic regression analyses revealed that combined analysis of mold specific T-cell frequencies and selected cytokine markers (A. fumigatus: IL-5 and TNF-α, R. arrhizus: IL-17A and IL-13) significantly improves classification performance, resulting in 75-90 % predictive power using 10-fold cross-validation. In conclusion, mold specific T-cell frequencies and their cytokine signatures offer promising potential in the assessment of environmental mold exposure. The cytokines identified in this pilot study should be validated in the clinical setting, e. g. in patients with hypersensitivity pneumonitis.

Photoinduced Liquefaction of Azobenzene-Containing Polymers.

Light can liquefy some solid azobenzene-containing polymers (azopolymers) by photoisomerization. Two types of photoinduced liquefaction of azopolymers have been reported: (1) polarized light can guide solid azopolymers "flow" along the polarization direction, which is called directional photofluidization and has been used for inscription of surface relief gratings (SRGs); (2) recently, some of us found that light can switch the glass transition temperatures (Tg ) of azopolymers and induce reversible solid-to-liquid transitions of these azopolymers. This Minireview demonstrates and compares directional photofluidization and photoinduced reversible solid-to-liquid transitions of azopolymers. Potential applications based on photoinduced liquefaction of azopolymers are highlighted and some remaining challenges in the field of photoinduced liquefaction of azopolymers are discussed.

Light-Switchable Azobenzene-Containing Macromolecules: From UV to Near Infrared.

Azobenzene-containing macromolecules (azo-macromolecules) such as azobenzene-containing polymers (azopolymers) and azobenzene-functionalized biomacromolecules are photoswitchable macromolecules. Trans-to-cis photoisomerization in conventional azo-macromolecules is induced by ultraviolet (UV) light. However, UV light cannot penetrate deeply into issue and has a very small fraction in sunlight. Therefore, conventional azo-macromolecules are problematic for biomedical and solar-energy-related applications. In this Feature Article, the strategies for constructing visible and near-infrared (NIR) light-responsive azo-macromolecules are reviewed, and the potential applications of visible- and NIR-light-responsive azo-macromolecules in biomedicine and solar energy conversion are highlighted. The remaining challenges in the field of photoswitchable azo-macromolecules are discussed.

Susceptibility of A. fumigatus-specific T-cell assays to pre-analytic blood storage and PBMC cryopreservation greatly depends on readout platform and analytes.

Mould-specific T cells detectable by flow cytometry or ELISPOT were proposed as a novel biomarker in invasive aspergillosis. To define protocols facilitating sample shipment and longitudinal analysis, this study evaluated the susceptibility of different functional assays for A. fumigatus-specific T-cell quantification and characterisation to pre-analytic delays. PBMCs from 6 healthy donors were analysed after immediate isolation, after 6 hours whole blood storage or after cryopreservation using 3 different common media. Functional responses to A. fumigatus lysate stimulation were comparatively assessed by flow cytometry, ELISPOT and 14-plex cytokine assay. After 6 hours pre-analytic storage, all functional assays showed reduced detection rates, higher coefficients of variation (CV) and widely varying individual recovery indices of specific T-cell response. While cryopreservation resulted in sufficient yields and largely unaltered cellular composition, outcomes of functional readouts significantly differed from freshly processed samples. For CD154-based flow cytometry, only cryopreservation in RPMI supplemented with autologous serum resulted in satisfactory detection rates and CVs. For ELISPOT and cytokine secretion assays, none of the cryopreservation protocols provided sufficient concordance with immediately processed samples. Even using the same readout platform, individual analytes widely varied in their susceptibility to cryopreservation, highlighting that distinct optimisation is required depending on the downstream assay.

Intra- and inter-individual variability of Aspergillus fumigatus reactive T-cell frequencies in healthy volunteers in dependency of mould exposure in residential and working environment.

Invasive aspergillosis remains a deadly disease in immunocompromised patients, whereas the combination of an exaggerated immune response and continuous exposure lead to various hyperinflammatory diseases. This pilot study aimed to gain an overview of the intra- and inter-individual variability in Aspergillus fumigatus reactive T-helper cells in healthy adults and the correlation with environmental mould exposure. In this flow cytometric study, the frequencies of CD154+ A. fumigatus reactive T cells were evaluated in 70 healthy volunteers. All subjects completed a standardised questionnaire addressing their mould exposure. Subjects with intensive mould exposure in their professional or residential surrounding demonstrated considerably higher mean frequencies of A. fumigatus reactive T-helper and T-memory cells. Comparative evaluation of multiple measurements over time demonstrated relatively conserved reactive T-cell frequencies in the absence of major changes to the exposure profile, whereas those frequently exposed in professional environment or with changes to their risk score demonstrated a marked dependency of antigen reactive T-cell frequencies on recent mould exposure. This pilot study was the first to provide data on the intra-individual variability in A. fumigatus reactive T-cell frequencies and its linkage to mould encounter. Fungus reactive T cells are to be considered a valued tool for the assessment of environmental mould exposure.

Hydrological controls on base metal precipitation and zoning at the porphyry-epithermal transition constrained by numerical modeling.

Ore precipitation in porphyry copper systems is generally characterized by metal zoning (Cu-Mo to Zn-Pb-Ag), which is suggested to be variably related to solubility decreases during fluid cooling, fluid-rock interactions, partitioning during fluid phase separation and mixing with external fluids. Here, we present new advances of a numerical process model by considering published constraints on the temperature- and salinity-dependent solubility of Cu, Pb and Zn in the ore fluid. We quantitatively investigate the roles of vapor-brine separation, halite saturation, initial metal contents, fluid mixing and remobilization as first-order controls of the physical hydrology on ore formation. The results show that the magmatic vapor and brine phases ascend with different residence times but as miscible fluid mixtures, with salinity increases generating metal-undersaturated bulk fluids. The release rates of magmatic fluids affect the location of the thermohaline fronts, leading to contrasting mechanisms for ore precipitation: higher rates result in halite saturation without significant metal zoning, lower rates produce zoned ore shells due to mixing with meteoric water. Varying metal contents can affect the order of the final metal precipitation sequence. Redissolution of precipitated metals results in zoned ore shell patterns in more peripheral locations and also decouples halite saturation from ore precipitation.

A Photopatternable Conjugated Polymer with Thermal-Annealing-Promoted Interchain Stacking for Highly Stable Anti-Counterfeiting Materials.

Photoresponsive polymers can be conveniently used to fabricate anti-counterfeiting materials through photopatterning. However, an unsolved problem is that ambient light and heat can damage anti-counterfeiting patterns on photoresponsive polymers. Herein, photo- and thermostable anti-counterfeiting materials are developed by photopatterning and thermal annealing of a photoresponsive conjugated polymer (MC-Azo). MC-Azo contains alternating azobenzene and fluorene units in the polymer backbone. To prepare an anti-counterfeiting material, an MC-Azo film is irradiated with polarized blue light through a photomask, and then thermally annealed under the pressure of a photonic stamp. This strategy generates a highly secure anti-counterfeiting material with dual patterns, which is stable to sunlight and heat over 200 °C. A key for the stability is that thermal annealing promotes interchain stacking, which converts photoresponsive MC-Azo to a photostable material. Another key for the stability is that the conjugated structure endows MC-Azo with desirable thermal properties. This study shows that the design of photopatternable conjugated polymers with thermal-annealing-promoted interchain stacking provides a new strategy for the development of highly stable and secure anti-counterfeiting materials.

A streamlined method for the fast and cost-effective detection of bacterial pathogens from positive blood cultures for the BacT/ALERT blood culture system using the Vitek MS mass spectrometer.

BACKGROUND AND OBJECTIVE: Prompt pathogen identification of blood stream infections is essential to provide appropriate antibiotic treatment. Therefore, the objective of this prospective single centre study was to establish an inexpensive, fast and accurate protocol for bacterial species identification with SDS protein-extraction directly from BacT/Alert® blood culture (BC) bottles by VitekMS®. RESULTS: Correct species identification was obtained for 198/266 (74.4%, 95%-CI = [68.8%, 79.6%]) of pathogens. The protocol was more successful in identifying 87/96 (91.4%, 95%-CI = [83.8%, 93.2%]) gram-negative bacteria than 110/167 (65.9%, 95%-CI = [58.1%, 73.0%]) gram-positive bacteria. The hands-on time for sample preparation and measurement was about 15 min for up to five samples. This is shorter than for most other protocols using a similar lysis-centrifugation approach for the combination of BacT/Alert® BC bottles and the Vitek® MS mass spectrometer. The estimated costs per sample were approx. 1.80€ which is much cheaper than for commercial kits. CONCLUSION: This optimized protocol allows for accurate identification of bacteria directly from blood culture bottles for laboratories equipped with BacT/Alert® blood culture bottles and VitekMS® mass spectrometer.

Photoresponsive metallopolymer nanoparticles for cancer theranostics.

Over the past decades, transition metal complexes have been successfully used in anticancer phototherapies. They have shown promising properties in many different areas including photo-induced ligand exchange or release, rich excited state behavior, and versatile biochemical properties. When encorporated into polymeric frameworks and become part of nanostructures, photoresponsive metallopolymer nanoparticles (MPNs) show enhanced water solubility, extended blood circulation and increased tumor-specific accumulation, which greatly improves the tumor therapeutic effects compared to low-molecule-weight metal complexes. In this review, we aim to present the recent development of photoresponsive MPNs as therapeutic nanomedicines. This review will summarize four major areas separately, namely platinum-containing polymers, zinc-containing polymers, iridium-containing polymers and ruthenium-containing polymers. Representative MPNs of each type are discussed in terms of their design strategies, fabrication methods, and working mechanisms. Current challenges and future perspectives in this field are also highlighted.

T-Cell Immune Surveillance in Allogenic Stem Cell Transplant Recipients: Are Whole Blood-Based Assays Ready to Challenge ELISPOT?

We compared the feasibility of 4 cytomegalovirus (CMV)- and Aspergillus-reactive T-cell immunoassay protocols in allogenic stem cell transplant recipients. While enzyme-linked immunospot performed best overall, logistically advantageous whole blood-based assays performed comparably in patients with less severe lymphocytopenia. CMV-induced interferon-gamma responses correlated strongly across all protocols and showed high concordance with serology.

Multiple Segmental Processes in Polymers with cis and trans Stereoregular Configurations.

Altering the stereochemistry of a single double bond in the side group of a polymer resulted in systems with unprecedented local dynamics. These include (i) the appearance of three segmental processes in the cis-polymers all with Vogel-Fulcher-Tammann (VFT) temperature dependence, (ii) the low steepness index associated with fragility, m, and (iii) the lowest pressure coefficient of Tg, dTg/dP, ever reported for polymers. We show that it is the inability of the cis-polymer to pack the side groups efficiently that controls the dynamics. Furthermore, the trans-polymers have the ability to crystallize. The wealth of dynamics reflects the cis/trans stereochemistry and the presence of different dipoles at specific positions sampling both the side group and backbone dynamics.

Mucorales spores induce a proinflammatory cytokine response in human mononuclear phagocytes and harbor no rodlet hydrophobins.

Mucormycoses are life-threatening infections in immunocompromised patients. This study characterizes the response of human mononuclear cells to different Mucorales and Ascomycota. PBMC, monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors were stimulated with resting and germinated stages of Mucorales and Ascomycota. Cytokine response and expression of activation markers were studied. Both inactivated germ tubes and resting spores of Rhizopus arrhizus and other human pathogenic Mucorales species significantly stimulated mRNA synthesis and secretion of proinflammatory cytokines. Moreover, R. arrhizus spores induced the upregulation of co-stimulatory molecules on moDCs and a specific T-helper cell response. Removal of rodlet hydrophobins by hydrofluoric acid treatment of A. fumigatus conidia resulted in enhanced immunogenicity, whereas the cytokine response of PBMCs to dormant R. arrhizus spores was not influenced by hydrofluoric acid. Scanning electron micrographs of Mucorales spores did not exhibit any morphological correlates of rodlet hydrophobins. Taken together, this study revealed striking differences in the response of human mononuclear cells to resting stages of Ascomycota and Mucorales, which may be explained by absence of an immunoprotective hydrophobin layer in Mucorales spores.

Photoswitching of glass transition temperatures of azobenzene-containing polymers induces reversible solid-to-liquid transitions.

The development of polymers with switchable glass transition temperatures (Tg) can address scientific challenges such as the healing of cracks in high-Tg polymers and the processing of hard polymers at room temperature without using plasticizing solvents. Here, we demonstrate that light can switch the Tg of azobenzene-containing polymers (azopolymers) and induce reversible solid-to-liquid transitions of the polymers. The azobenzene groups in the polymers exhibit reversible cis-trans photoisomerization abilities. Trans azopolymers are solids with Tg above room temperature, whereas cis azopolymers are liquids with Tg below room temperature. Because of the photoinduced solid-to-liquid transitions of these polymers, light can reduce the surface roughness of azopolymer films by almost 600%, repeatedly heal cracks in azopolymers, and control the adhesion of azopolymers for transfer printing. The photoswitching of Tg provides a new strategy for designing healable polymers with high Tg and allows for control over the mechanical properties of polymers with high spatiotemporal resolution.

Geologic controls on supercritical geothermal resources above magmatic intrusions.

A new and economically attractive type of geothermal resource was recently discovered in the Krafla volcanic system, Iceland, consisting of supercritical water at 450 °C immediately above a 2-km deep magma body. Although utilizing such supercritical resources could multiply power production from geothermal wells, the abundance, location and size of similar resources are undefined. Here we present the first numerical simulations of supercritical geothermal resource formation, showing that they are an integral part of magma-driven geothermal systems. Potentially exploitable resources form in rocks with a brittle-ductile transition temperature higher than 450 °C, such as basalt. Water temperatures and enthalpies can exceed 400 °C and 3 MJ kg(-1), depending on host rock permeability. Conventional high-enthalpy resources result from mixing of ascending supercritical and cooler surrounding water. Our models reproduce the measured thermal conditions of the resource discovered at Krafla. Similar resources may be widespread below conventional high-enthalpy geothermal systems.