Stimulation of pDC by PPVO is greatly diminished in the absence of TLR9.

<p>BMDC of WT and TLR9^−/− mice were generated and purified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106188#s2" target="_blank">material and methods</a>. Purified pDC were stimulated in triplicates with the indicated stimuli and supernatants and cells were harvested after 24 h. Cytokines were determined in culture supernatants using ELISA and expression of CD86 by flow cytometry. One representative of three independent experiments is depicted for cytokine data. Histograms of one representative and pooled data of three independent experiments are shown for CD86 expression. Cytokine concentrations and CD86 expression were statistically analyzed using 2-way ANOVA and Bonferroni post-test, and significant differences between WT and TLR9^−/− pDC responses are indicated by asterisks.</p

PPVO sensing requires endosomal maturation in pDC but not in cDC.

<p>BMDC were generated in the presence of Flt3-ligand and pDC and cDC purified by FACS as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106188#s2" target="_blank">material and methods</a>. Purified pDC and cDC were stimulated in triplicates as indicated for 24 h in the absence or presence of chloroquine and supernatants were analysed for the indicated cytokines. Concentrations of cytokines are presented as mean +/− SEM. One representative experiment of three is presented for cytokine data. Remaining cells were stained and analysed for the expression of co-stimulatory CD86 presented as mean +/− SEM of the median fluorescence intensity. Pooled data of two experiments are shown for CD86 expression. Statistically significant differences between stimulations in the absence and presence of chloroquine were determined by 2-way ANOVA and Bonferroni post-test and are indicated by asterisks.</p

PPVO-induced pDC activation is blocked by TLR9-specific iCpG-ODN.

<p>Purified BM-pDC were stimulated in triplicates as indicated for 24 h in the absence or presence of iCpG-ODN (iCpG) and supernatants were analysed for the indicated cytokines. Concentrations of cytokines are presented as mean +/− SEM. One representative experiment of three is presented for cytokine data. Remaining cells were stained and analysed for the expression of co-stimulatory CD86 presented as the mean +/− SEM of the median fluorescence intensity. Pooled data of three experiments are shown for CD86 expression. Statistically significant differences between stimulations in the absence and presence of iCpG-ODN (5 µM) were determined by 2-way ANOVA and Bonferroni post-test and are indicated by asterisks.</p

Data_Sheet_1_Evaluation of proline-rich antimicrobial peptides as potential lead structures for novel antimycotics against Cryptococcus neoformans.docx

BackgroundCryptococcosis and cryptococcal meningitis, caused by Cryptococcus neoformans infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against C. neoformans. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment.ResultsA preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the C. neoformans strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 μmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.</p

Early fungal growth control in pulmonary infection with <i>C. neoformans</i> in the presence of IL-4Rα signaling.

<p>Wild-type (WT, open circle) and IL-4Rα^−/− (gray circle) mice on C57BL/6J background were infected intranasally with <i>C. neoformans</i>. Analysis of fungal burdens in the lung was done at different days <i>post infectionem</i> (dpi) as indicated. Shown is data from n = 7 mice per group from one representative of three independent experiments (14 dpi) or from two independent experiments (7; 21 and 42 dpi). Statistical analysis was done using the unpaired Student's t-test (7 dpi) or Mann-Whitney test. *P<0.05; **P<0.01.</p

In the presence of IL-4Rα elevated pulmonary chemokine expression, IFN-γ mRNA expression and NO production.

<p>Following infection, WT (open circle) and IL-4Rα^−/− (gray circle) mice were sacrificed at the time points indicated (A, C) or at 14 days after infection (B). RT-qPCR analyses were done to determine the expression of mRNAs as indicated. Data are derived from one (A, C, 0 and 7 dpi) or one representative out of two (A, 14 dpi) or three (C, 14 dpi) experiments (n = 6–7 mice per genotype and experiment). Statistical analysis was done using the unpaired Student's t-test (ns, not significant; *P<0.05; **P<0.01; ***P<0.001 (A, C)). The concentration of nitric oxide (NO) in cell culture supernatants was determined using the Griess reaction. Pooled data from two different experiments are shown. Dotted line represents detection limit. Statistical analysis was done using the Mann-Whitney test. **P<0.01 (B).</p

Stronger pulmonary inflammation, eosinophilia, and mucus production in WT as compared with IL-4Rα^−/− mice.

<p>Lung slices from WT and IL-4Rα^−/− mice infected for 14 days were stained with H&E (A-F) and periodic acid Schiff reagent (G, H). Leukocyte infiltration and fungal load are depicted in panels A, C and B, D. Sites of inflammation contain eosinophils (arrowheads) and large, multinucleated macrophages (E) or lymphocytes (F). Mucus production by bronchial epithelial cells is depicted in G and H. One representative experiment out of three with n = 6–7 animals per group is shown.</p

Additional file 1 of Tissue S100/calgranulin expression and blood neutrophil-to-lymphocyte ratio (NLR) in dogs with lower urinary tract urothelial carcinoma

Additional file 1: Supplementary Table 1. Overview of the patient demographic, treatment, outcome, and study participation information for the dogs in-cluded in the study (n=55)

Api88 Is a Novel Antibacterial Designer Peptide To Treat Systemic Infections with Multidrug-Resistant Gram-Negative Pathogens

The emergence of multiple-drug-resistant (MDR) bacterial pathogens in hospitals (nosocomial infections) presents a global threat of growing importance, especially for Gram-negative bacteria with extended spectrum β-lactamase (ESBL) or the novel New Delhi metallo-β-lactamase 1 (NDM-1) resistance. Starting from the antibacterial peptide apidaecin 1b, we have optimized the sequence to treat systemic infections with the most threatening human pathogens, such as <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, <i>Pseudomonas aeruginosa</i>, and <i>Acinetobacter baumannii</i>. The lead compound Api88 enters bacteria without lytic effects at the membrane and inhibits chaperone DnaK at the substrate binding domain with a <i>K</i>_D of 5 μmol/L. The Api88-DnaK crystal structure revealed that Api88 binds with a seven residue long sequence (PVYIPRP), in two different modes. Mice did not show any sign of toxicity when Api88 was injected four times intraperitoneally at a dose of 40 mg/kg body weight (BW) within 24 h, whereas three injections of 1.25 mg/kg BW and 5 mg/kg BW were sufficient to rescue all animals in lethal sepsis models using pathogenic <i>E. coli</i> strains ATCC 25922 and Neumann, respectively. Radioactive labeling showed that Api88 enters all organs investigated including the brain and is cleared through both the liver and kidneys at similar rates. In conclusion, Api88 is a novel, highly promising, 18-residue peptide lead compound with favorable <i>in vitro</i> and <i>in vivo</i> properties including a promising safety margin