Effect of electric field polarization and temperature on the effective permittivity and conductivity of porous anodic aluminium oxide membranes

Porous insulators offer new opportunities for the controlled guest–host synthesis of nanowires for future integrated circuits characterized by low propagation delay, crosstalk and power consumption. We propose a method to estimate the effect of the electric field polarization and temperature on the electrical properties of different types of synthesized porous anodic aluminium oxide membranes. It results that the effective permittivity along the pore axis is generally 20% higher than the one in the orthogonal direction. The type of solution and the voltage level applied during anodization are the main parameters affecting the AAO templates characteristics, i.e. their porosity and chemical content. The values of permittivity of the final material, are typically in the range 2.6–3.2 for large pore diameter membranes including phosphorus element and having a low water content, and in the range 3.5–4 for the ones with smaller pores, and showing sulphur element incorporation. Moreover, the dc conductivity of the different membranes appears to be correlated to the pore density

Human hantavirus infection elicits pronounced redistribution of mononuclear phagocytes in peripheral blood and airways

Hantaviruses infect humans via inhalation of virus-contaminated rodent excreta. Infection can cause severe disease with up to 40% mortality depending on the viral strain. The virus primarily targets the vascular endothelium without direct cytopathic effects. Instead, exaggerated immune responses may inadvertently contribute to disease development. Mononuclear phagocytes (MNPs), including monocytes and dendritic cells (DCs), orchestrate the adaptive immune responses. Since hantaviruses are transmitted via inhalation, studying immunological events in the airways is of importance to understand the processes leading to immunopathogenesis. Here, we studied 17 patients infected with Puumala virus that causes a mild form of hemorrhagic fever with renal syndrome (HFRS). Bronchial biopsies as well as longitudinal blood draws were obtained from the patients. During the acute stage of disease, a significant influx of MNPs expressing HLA-DR, CD11c or CD123 was detected in the patients' bronchial tissue. In parallel, absolute numbers of MNPs were dramatically reduced in peripheral blood, coinciding with viremia. Expression of CCR7 on the remaining MNPs in blood suggested migration to peripheral and/or lymphoid tissues. Numbers of MNPs in blood subsequently normalized during the convalescent phase of the disease when viral RNA was no longer detectable in plasma. Finally, we exposed blood MNPs in vitro to Puumala virus, and demonstrated an induction of CCR7 expression on MNPs. In conclusion, the present study shows a marked redistribution of blood MNPs to the airways during acute hantavirus disease, a process that may underlie the local immune activation and contribute to immunopathogenesis in hantavirus-infected patients

Susceptibility of classical monocytes and CD1c⁺ MDCs to PUUV <i>in vitro</i>.

<p>(<b>A</b>) Human CM (top panel, green) and CD1c⁺ MDCs (bottom panel, coral) were isolated from peripheral blood of healthy volunteers. Flow cytometry dot plots show live, HLA-DR⁺ CD11c⁺ CD14⁺ CD16^- monocytes or live, CD11c⁺ CD1c⁺ MDCs. Numbers in gate depict the frequency of cells out of total live cells. One representative donor of six is shown. Cells were left unexposed, exposed to PUUV or UV-inactivated PUUV for 2 h at an MOI of 7.5. Cells were washed and subsequently incubated for 12–60 h. (<b>B</b>) Immunofluorescence staining with patient serum on CM (left panel) and CD1c⁺ MDCs (right panel) on uninfected and PUUV-infected cells 40 h after infection indicates detectable viral antigen (green). Cells were counterstained with DAPI (gray) and also stained for HLA-DR (blue). Scale bar, 10 μm. (<b>C</b>) Bar graphs summarize the mean±SD of PUUV⁺ CM (left panel, n = 3) or CD1c⁺ MDCs (n = 3). n.d., not detectable. (<b>D</b>) Relative expression of PUUV RNA was measured in CM and CD1c⁺ MDCs (n = 2) after 60 h of infection. Bar graphs show 2^-ΔCt values relative to the housekeeping gene <i>β-Actin</i>. (<b>E</b>) Viability of cells was assessed by flow cytometry based on a LIVE/DEAD dye. Graphs show mean±SD viability of CM (top panel, n = 4) or CD1c⁺ MDC (bottom panel, n = 6). (<b>F</b>) Histograms indicate changes in expression of CCR2, CCR4, CCR6 and CD86 in cells exposed to PUUV (ocean blue) compared to unexposed (black line) from one representative donor of CM (n = 4) and CD1c⁺ MDCs (n = 6). Fluorescence minus one (FMO) controls are shown in gray. <b>(G</b>) Bar graphs summarize the MFI±SD of CCR2, CCR4, CCR6 and CD86 (n = 4–6) in cells left unexposed (white), exposed to PUUV (ocean blue) or UV PUUV (patterned ocean blue). <b>(H</b>) Flow cytometry dot plots show changes in CCR7 in cells exposed to PUUV or unexposed. Numbers in gate depict the frequency of CCR7⁺ cells out of total CM (n = 4) or CD1c⁺ MDC (n = 6). (<b>I</b>) Bar graphs summarize the MFI±SD of CCR7 (n = 4–6). Statistical differences were assessed using paired <i>t</i>-test: * <i>p</i><0.05, n.s. not significant.</p

Decreased numbers of CD1c⁺ and CD141⁺ MDCs in blood during acute HFRS.

<p>(<b>A</b>) Gating strategy for the identification of CD1c⁺ MDCs (coral) and CD141⁺ MDCs (maroon) by flow cytometry after gating out lineage (CD3, CD20, CD56) cells, gating on HLA-DR⁺, CD11c⁺ cells, and gating out monocytes (CD14, CD16). Representative flow cytometry plots from one representative UC and HFRS patient are shown. (<b>B</b>) Mean±SD absolute numbers of MDC populations were evaluated in longitudinal samples in HFRS patients in comparison to UC. (<b>C</b>) Graphs show detectable viral load in plasma measured by viral RNA quantification from one representative HFRS patient. Virological data (left axis; black line) and absolute numbers of MDCs (right axis; colored line) plotted over time from one representative HFRS patient. Differences in mean absolute number of MDCs were assessed using Poisson regression: ** <i>p</i><0.01, *** <i>p</i><0.001, n.s. not significant.</p

Upregulation of CCR7 on MNPs in blood of HFRS patients during acute infection.

<p>(<b>A</b>) Representative flow cytometry plots showing CCR7 expression on CD1c⁺ MDCs from one representative UC and one HFRS patient are shown. (<b>B</b>) Mean±SD frequencies of CCR7⁺ CMs (green), IM (red), NCM (blue), CD1c⁺ MDCs (coral), CD141⁺ MDCs (maroon) and PDCs (teal) were quantified in longitudinal samples from HFRS patients in comparison to UC. (<b>C</b>) Histograms indicate upregulation of CD70 on CM, and upregulation of CD86 and CD70 on CD1c⁺ MDCs of a HFRS patient during the acute phase. (<b>D</b>) Bar graphs summarize the MFI±SD of CD70 and CD86 in CCR7⁺ and CCR7^- CM and CD1c⁺ MDCs. Differences in proportion of CCR7⁺ cells were assessed using logistic regression; * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001, n.s. not significant.</p

Decreased number of PDCs in blood during acute HFRS.

<p>(<b>A</b>) Gating strategy for the identification of PDCs (teal) by flow cytometry after gating out lineage (CD3, CD20, CD56) cells, and gating on HLA-DR⁺ CD11c^- cells. Representative flow cytometry plots from one representative UC and HFRS patient are shown. (<b>B</b>) Mean±SD absolute numbers of PDCs were evaluated in longitudinal samples in HFRS patients in comparison to UC. (<b>C</b>) Graphs show detectable viral load in plasma measured by viral RNA quantification from one representative HFRS patient Virological data (left axis; black line) and absolute numbers of PDCs (right axis; colored line) are shown over time from one representative HFRS patient. Differences in mean absolute number of PDCs were assessed using Poisson regression: *** <i>p</i><0.001, n.s. not significant.</p

Respiratory involvement in hantavirus-infected patients.

<p><b>(A)</b> Hantavirus-infected patients diagnosed with hemorrhagic fever with renal syndrome (HFRS patients) underwent bronchoscopy (median day 9, n = 17) for collection of endobronchial biopsies and bronchoalveolar lavage (BAL). Multiple blood draws were taken from 12 patients during acute (median day 6, total of all patients = 28) and convalescent (median day 89, total of all patients = 30) phases. Uninfected controls (UC) were sampled for blood (n = 12) and endobronchial biopsies (n = 16). <b>(B)</b> Detectable viral load in BAL cells of 15 patients were further classified according to whether they experienced respiratory symptoms (left panel) or needed oxygen treatment (right panel). Bar graphs show mean±SD. <b>(C)</b> Mean±SD absolute numbers of CD8⁺ T cells were evaluated by flow cytometry in BAL of patients with or without oxygen treatment. <b>(D)</b> Granzyme B was measured in BAL fluid of patients with (n = 5) or without (n = 12) oxygen treatment and presented as mean±SD. <b>(E)</b> Representative images of CD8 staining in endobronchial biopsies from UC (n = 16) and HFRS patient (n = 17). Specific staining appears in red, and cell nuclei are counterstained with hematoxylin in blue. Arrows indicate positive staining. Visualization was performed using immunohistochemistry. Scale bar, 50 μm. <b>(F)</b> Bar graph summarizes mean±SD comparing HFRS patients (n = 17) during the acute phase of infection with UC (n = 16). The number of CD8⁺ cells is expressed as cells/mm². Statistical differences were assessed using two-tailed Mann–Whitney U-test: * <i>p</i><0.05, n.s. not significant.</p

Infiltration of MNPs into the airways during acute hantavirus infection.

<p>Endobronchial biopsies were taken from patients with acute HFRS and age- and sex-matched UC. (<b>A</b>) Representative images of endobronchial biopsies revealing high numbers of HLA-DR⁺ cells in HFRS patients (n = 5), as compared to UC (n = 5) are shown. Specific staining appears in red, and cell nuclei are counterstained with hematoxylin in blue. Arrows indicate positive staining. Visualization was performed using immunohistochemistry. Scale bar, 100 μm. (<b>B</b>) Bar graph summarizes mean±SD percentage of HLA-DR stained areas of all subjects. Representative images of (<b>C</b>) CD11c and (<b>E</b>) CD123 staining in lamina propria and epithelium in endobronchial biopsies from HFRS patients and UC. Scale bar, 50 μm. (<b>D</b> and <b>F</b>) Bar graphs summarize mean±SD comparing HFRS patients (n = 17) during the acute phase of infection with UC (n = 16). The numbers of CD11c⁺ or CD123⁺ cells are expressed as cells/mm² of epithelium and lamina propria, respectively. Statistical differences were assessed using two-tailed Mann–Whitney U-test: * <i>p</i><0.05 ** <i>p</i><0.01. Graphs show the positive correlation (ρ) of CD8⁺ cells to (<b>G</b>) CD11c⁺ cells and (<b>H</b>) CD123⁺ cells in endobronchial biopsies of HFRS patients as assessed by Spearman’s correlation test (n = 17). (<b>I</b>) Detectable viral load in BAL cells (per 10⁴ cells) and plasma (per mL) of HFRS patients measured by viral RNA quantification on the day of bronchoscopy. Lines indicate paired measurements in BAL cells and plasma of individual patients (n = 8).</p