Synergistic effects of TLR ligands with poke weed lectin.

<p><b>A: Effect of TLR2 blockage on human B cell proliferation in response to PWM.</b> CD19+ human peripheral blood B cells were labelled with CFSE, preincubated with anti-TLR2 mAb or the corresponding isotype control (murine IgG_1κ), and stimulated with Pam₃CSK₄ or PWM (10 µg/ml) ± SpA (5 µg/ml) for 5 days. Proliferation was assessed by CFSE dilution. The gate denotes the percentage of live gated proliferating B cells as depicted. One representative experiment of n = 3 is shown. <b>B: Synergistic effects of PWM and immunostimulatory DNA.</b> Human B cells were stained with CFSE for assessment of B cell proliferation. Stimulation was performed with CpG ODN 2006 (PTO), DNAse- or mock (reaction buffer only)-treated PWM, 2006 GC (PTO) or CpG 2006 (PO) and combinations thereof as indicated. The graphs show the results from one representative experiment from n≥3. The percentage of proliferating B cells is indicated in the graphs.</p

TLR-dependency of Poke weed mitogen (PWM)-induced cell stimulation.

<p><b>A: B cell proliferation.</b> Human CD19+ peripheral blood B cells were stained with CFSE and stimulated with 1 or 10 µg/ml PWM in the presence or absence of 5 µg/ml <i>Staphylococcus aureus</i> protein A (SpA) or 5 µg/ml anti-human Ig F(ab′)₂ fragments (aIg) as B cell receptor stimuli, with SpA or aIg alone, or left unstimulated. After 5 days B cells were harvested, stained with anti-CD20 and live gated cells were analyzed for CFSE dilution. The percentages of live gated cells (upper right corner) and proliferating cells (left) are indicated in each diagram. The diagrams depict the results from one representative experiment of n = 3. <b>B: TNF-induction.</b> Human PBMC were stimulated for four hours with or without PWM, TLR2 ligands FSL-1R (FSL) and Pam₃CSK₄ (P3), phosphorothioate-modified DNA ODN CpG 2006 (CpG) and 2006 GC (GC) and LPS in the presence of Brefeldin A (BfA) or with LPS in the absence of BfA. Subsequently, intracellular staining was performed with an anti-human TNF mAb and TNF expression was analyzed by flow cytometry. The results show a summary of the data obtained in n = 4 experiments. Mean values of anti-TNF mean fluorescence intensities are provided ± SEM. <b>C: Antagonization with Polymyxin B.</b> CFSE-stained human CD19+ B cells were stimulated with PWM in the presence and absence of polymyxin B. Proliferation was assessed by flow cytometric analysis of CFSE dilution on day 5. The results obtained in two representative donors of n = 3 are shown. <b>D: MyD88-dependency of B cell stimulation.</b> B220+ B cells were isolated from the spleens of MyD88^−/− mice and their wild type counterparts. B cells were stimulated with CpG, P3, PWM and PMA/Ionomycin (P/I) and harvested after 72 hours and a 18 hour pulse with ³H-thymidine. The diagram shows the average values in counts per minute (cpm) of n = 4 experiments ± SEM. <b>E: TLR-dependency.</b> Wild type, TLR2−/− and TLR9−/− B cells were isolated from murine spleen with anti-CD19 microbeads, labelled with CFSE and stimulated with TLR2 ligands Pam₃CSK₄ (P3) and FSL-1R (FSL), PWM (10 µg/ml), TLR9 ligand CpG ODN 1668 or 1668 GC control ODN. After 4 days B cell proliferation (CFSE dilution) was quantified by flow cytometry. The diagram depicts the mean fluorescence intensity (MFI) for CFSE in live gated cells as mean value ± SEM from n = 4 experiments. Note that low MFI corresponds to strong proliferation while high MFI values reveal absence of proliferation.</p

Detection of TLR ligands in PWM preparations.

<p><b>A:</b> Human B cells were stimulated with different lots of PWM. Proliferation rates were quantified by 3H-thymidine incorporation (cpm = counts per minute). The diagram shows the mean values ± SEM from n = 3 experiments. <b>B:</b> LAL-assay was performed to quantify the LPS content in PWM Lot. A–D. The diagram depicts the mean values ± standard deviation obtained by testing in quadruplicates. <b>C:</b> TLR2 activity was assessed by measuring IL-8 concentrations in the supernatants of pTLR2-transfected or non-transfected HEK293 cells stimulated with different lots of PWM (left) or Pam₃CSK₄ (right) at the concentrations indicated. One representative experiment performed in triplicates of n = 2 is shown.</p

Contribution of the lectin component.

<p><b>A:</b> Human B cell proliferation in response to PWM (Lot. B and C) in the presence or absence of N,N-di-acetylchitobiose or N,N,N-tri-acetylchitotriose was assessed by ³H-thymidine incorporation given in counts per minute (cpm). The mean values ± SEM from one representative experiment performed in triplicates of n = 2 is shown. <b>B:</b> Human CD19+ B cells were left unstimulated or stimulated with 0.25 µM GpC PTO ODN (GC) and/or 10 µg/ml of PWM, 5 µg/ml SpA or 10 µg/ml anti-Ig (aIg) for 72 hours. Proliferation was quantified by ³H-thymidine incorporation. The diagram shows the means from n = 4 experiments ± SEM. The values obtained were normalized to GC = 1 (710±280 cpm = mean ± SEM). <b>C:</b> Human CD19+ B cells were stained with CFSE and stimulated with highly purified PWM (Lot. D) or <i>Lycopersicon esculentum</i> lectin (LEA) with or without GpC PTO ODN (GC). Proliferation was quantified by CFSE dilution on day 4. The percentage of proliferating cells is provided in each dot plot. The results from two independent donors of n = 4 are shown.</p

Contribution of TLR ligands to B cell activation.

<p><b>A: Stimulation of human B cells with TLR ligands.</b> CFSE-labelled CD19+ human B cells were stimulated with or without TLR ligands (TLR2 ligand MALP-2, TLR4 ligand LPS (0.1 and 1 µg/ml) or TLR9 ligand CpG ODN 2006 (CpG)) and/or BCR stimuli (5 µg/ml SpA or anti-Ig (aIg)). After 5 days cells were harvested, stained with anti-CD20 and analyzed by flow cytometry. The graphs depict cell survival (percentage of live gated cells; upper right angle) and cell proliferation (CFSE dilution; % proliferating cells of live gated cells (left) where indicated). The experiment shown is representative of n≥3 experiments. <b>B–D: Assessment of TLR2 activity in PWM preparations.</b> HEK293 cells were transfected with pTLR2 or lipofectamine alone (Lf). <b>B:</b> non-transfected and pTLR2-transfected HEK293 cells were stained for TLR2 expression with anti-TLR2 mAb or the respective isotype control as indicated. <b>C:</b> HEK293 cells transfected with pTLR2 or Lf only were stimulated with Pam₃CSK₄ (P3) or PWM (10 µg/ml). After 24 hours cellular supernatants were collected and analyzed for IL-8 secretion. One representative experiment of n≥3 experiments is shown. <b>D:</b> HEK293 cells transfected with pTLR2 were pretreated with anti-TLR2 mAb or the isotype control before stimulation with Pam₃CSK₄ (P3) or PWM (10 µg/ml). IL-8 was quantified in the 24 hour supernatants. <b>E: 16S rDNA PCR.</b> DNA isolation and PCR amplification of bacterial 16S ribosomal DNA from DNA from <i>E. coli</i> (EC), a negative clinical specimen (NC), a positive clinical sample (CS) and the PWM preparation (PW) or the water control (H₂O), with an expected PCR fragment size of approximately 900 bp.</p

βLACTA^TM, VITEK2 and PHOENIX100 results in the strain collective.

<p>βLACTA^TM, VITEK2 and PHOENIX100 results in the strain collective.</p

Antigen delivery to dendritic cells shapes human CD4⁺ and CD8⁺ T cell memory responses to <i>Staphylococcus aureus</i>

<div><p>Intracellular persistence of <i>Staphylococcus aureus</i> favors bacterial spread and chronic infections. Here, we provide evidence for the existence of human CD4⁺ and CD8⁺ T cell memory against staphylococcal antigens. Notably, the latter could provide a missing link in our understanding of immune control of intracellular <i>S</i>. <i>aureus</i>. The analyses showed that pulsing of monocyte-derived dendritic cells (MoDC) with native staphylococcal protein antigens induced release of Th2-associated cytokines and mediators linked to T regulatory cell development (G-CSF, IL-2 and IL-10) from both CD4⁺ and CD8⁺ T cells, thus revealing a state of tolerance predominantly arising from preformed memory T cells. Furthermore, G-CSF was identified as a suppressor of CD8⁺ T cell-derived IFNγ secretion, thus confirming a tolerogenic role of this cytokine in the regulation of T cell responses to <i>S</i>. <i>aureus</i>. Nevertheless, delivery of <i>in vitro</i> transcribed mRNA-encoded staphylococcal antigens triggered Th1-biased responses, e.g. IFNγ and TNF release from both naïve and memory T cells. Collectively, our data highlight the potential of mRNA-adjuvanted antigen presentation to enable inflammatory responses, thus overriding the existing Th2/Treg-biased memory T cell response to native <i>S</i>. <i>aureus</i> antigens.</p></div

Species-specific distribution of ESBL, AmpC, K1 and KPC in this study.

<p>Species-specific distribution of ESBL, AmpC, K1 and KPC in this study.</p

Sensitivity and specificity for βLACTA^TM for 3-GCR, ESBL and AmpC in <i>Enterobacteriaceae</i> and for ESBL detection in <i>E</i>. <i>coli</i> and <i>Klebsiella</i> spp.

<p>Sensitivity and specificity for βLACTA^TM for 3-GCR, ESBL and AmpC in <i>Enterobacteriaceae</i> and for ESBL detection in <i>E</i>. <i>coli</i> and <i>Klebsiella</i> spp.</p

T cell cytokine profiles in response to <i>spa</i> mRNA-encoded and SpA protein delivered antigens.

<p>Cytokine secretion profiles in day 5 supernatants of CD4⁺ (left panel) or CD8⁺ T cells (right panel) stimulated with <i>spa</i> mRNA or SpA protein antigens was performed using a multiplex cytokine array: <b>(a)</b> Th1 cytokines (IFNγ, TNF) and <b>(b)</b> Th2 cytokines (IL-5, IL-13). The graphs depict the mean values ± SEM obtained from n = 6 independent donors. Experiments were carried out in duplicates. For Th1 cytokines, one-way ANOVA was used to test significance of multiple conditions; for Th2 cytokines, p values refer to condition with <i>spa</i> mRNA (paired student’s t-test; p**<0.01, p*< 0.05).</p