Participants Profile.

<p>Participants Profile.</p

Leukemia cell signaling and signaling of non-malignant B-cells

Leukemia cell signaling Acute leukemia (AL) is the most common pediatric cancer. Approximately 90 - 100 children is diagnosed every year in the Czech Republic. Acute leukemia is a complex disease that is pathologically manifested at the DNA, mRNA, protein and cellular level. Leukemic cells aberrantly express molecules that are found in other cell types under physiological conditions and their functional involvement in leukemic cells is unknow. We found that aberrantly expressed CEACAM6 increases the expression and affinity of integrins, increases the phosphorylation of intracellular kinases Akt, p38MAPK and p44/42 MAPK and triggers apoptosis in B- cell precursor acute lymphoblastic leukemia cells. Adaptor molecule NTAL, aberrantly expressed in T-cell acute lymphoblastic leukemia, signals through intracellular kinase p44/42 MAPK and potentiates corticosteroid induced apoptosis. Current leukemia research is focused mainly on monitoring of mutations at the DNA level, however, the functional consequences of these changes on cellular machineries are not straightforward. Since proteome analysis can provide link between gene sequence and cellular physiology, proteomics will contribute to elucidate mechanism of disease, prognosis and response to treatment. Protein microarrays technology is of major..

Phylogenetic analyses.

<p>Phylogenetic tree for the HA gene (AA based) as well as the results on the genetic analyses of the NA gene, antigenic typing results and information on the time of sample collection of each virus; viruses in colour and framed by red rectangles indicate reference viruses; viruses in bold and with * indicate vaccinated influenza positive cases; viruses in red and bold indicate influenza B viruses with HA and NA from distinct influenza B lineages. (4A) detailed results of 59 influenza A(H1N1)pdm09 viruses, (4B) detailed results of 98 A(H3N2) viruses, and (4C) detailed results of 58 influenza B viruses.</p

Inclusion and exclusion criteria.

<p>Inclusion and exclusion criteria applied to the data set used for VE estimates.</p

Vaccine effectiveness estimates.

<p>Vaccine effectiveness estimates.</p

The Role of Alveolar Epithelial Type II-Like Cells in Uptake of Structurally Different Antigens and in Polarisation of Local Immune Responses

<div><p>Background</p><p>Our previous studies on intranasal tolerance induction demonstrated reduction of allergic responses with different allergen constructs. The underlying mechanisms varied depending on their conformation or size.</p><p>Objective</p><p>The aim of the present study was to compare the uptake of two structurally different allergen molecules within the respiratory tract following intranasal application.</p><p>Methods</p><p>The three-dimensional Bet v 1 (Bv1-Protein) and the T cell epitope peptide of Bet v 1 (Bv1-Peptide) were labelled with 5,6-Carboxyfluorescein (FAM) and their uptake was investigated in lung cells and cells of the nasal associated lymphoid tissue from naive and sensitised BALB/c mice. Phenotypic characterisation of FAM⁺ lung cells after antigen incubation <i>in vitro</i> and after intranasal application was performed by flow cytometry. Impact of Bv1-Protein and Bv1-Peptide on cytokine profiles and gene expression <i>in vivo</i> or in an alveolar epithelial type II (ATII) cell line were assessed in mono- and co-cultures with monocytes using ELISA and quantitative real-time PCR.</p><p>Results</p><p>Both antigens were taken up preferably by ATII-like cells (ATII-LCs) in naive mice, and by macrophages in sensitised mice. After intranasal application, Bv1-Peptide was taken up faster and more efficiently than Bv1-Protein. <i>In vivo</i> and <i>in vitro</i> experiments revealed that Bv1-Protein induced the transcription of thymic stromal lymphopoietin mRNA while Bv1-Peptide induced the transcription of IL-10 and MCP1 mRNA in ATII-LCs.</p><p>Conclusion and Clinical Relevance</p><p>Both tested antigens were taken up by ATII-LCs under steady state conditions and induced different polarisation of the immune responses. These data may have an important impact for the generation of novel and more effective prophylactic or therapeutic tools targeting the respiratory mucosa.</p></div

Time-dependent uptake of Bv1-Protein and Bv1-Peptide in NALT and lungs and phenotypic characterisation of FAM⁺ lung celIs <i>in vivo</i>.

<p>20 μg of Bv1-Protein-FAM or Bv1-Peptide-FAM were intranasal administered to naive BALB/c mice (n = 3 per time point). (<b>A</b> and <b>B</b>) After 1, 6, 24, and 48 hours NALT and lungs were harvested, and analysed by flow cytometry. Dead cells were identified via 7-AAD staining and excluded from analysis. Data are the pool from three independently performed experiments of identical design. (<b>C</b>) FAM^<b>+</b> cells were gated and antigen uptake capacity of macrophages (CD11b^<b>+</b>/CD11c^<b>-</b>), dendritic cells (CD11b^<b>-</b>/CD11c^<b>+</b>), B cells (B220^<b>+</b>/CD19^<b>+</b>), and ATII-LCs (CD11b^<b>-</b>/CD11c^<b>-</b>/CD16/32^<b>-</b>/CD19^<b>-</b>/CD31^<b>-</b>/CD45^<b>-</b>/F4/80^<b>-</b>/MHCII^<b>+</b>) in lungs of naive mice was investigated by flow cytometry. Data are the pool of two independently performed experiments of identical design. Values represent means ± SEM. A value P<0.05 was considered to be significant. *P<0.05 and ***P<0.001 indicate levels significantly different from time point 0 hours. ATII-LCs = ATII-like cells; DCs = dendritic cells; Mϕ = macrophages; NALT = nasal associated lymphoid tissue.</p

Uptake of Bv1-Protein and Bv1-Peptide in lungs of naive and sensitised mice <i>in vivo</i>.

<p>(<b>A</b>) Experimental design: Mice were sensitised 3 times intraperitonealy with 1 μg of Bet v 1 on days 0, 14 and 28. Intranasal challenge with 100 μg of birch pollen extract was performed one week after last sensitisation on days 35, 36 and 37. On day 40, 20 μg of Bv1-Protein-FAM and Bv1-Peptide-FAM were intranasal administered to naive and allergic BALB/c (n = 3) mice and lungs were collected after 1 (for Bv1-Peptide) or 6 hours (for Bv1-Protein). Dead cells were identified via 7-AAD staining and excluded from analysis. (<b>B</b> and <b>C</b>) FAM^<b>+</b> cells were gated and antigen uptake capacity of macrophages (CD11b^<b>+</b>/CD11c^<b>-</b>), dendritic cells (CD11b^<b>-</b>/CD11c^<b>+</b>), B cells (B220^<b>+</b>/CD19^<b>+</b>), and ATII-LCs (CD11b^<b>-</b>/CD11c^<b>-</b>/CD16/32^<b>-</b>/CD19^<b>-</b>/CD45^<b>-</b>/F4/80^<b>-</b>) in lungs of naive and allergic mice was investigated by flow cytometry. Data are the pool from three independently performed experiments of identical design. Values represent means ± SEM. A value P<0.05 was considered to be significant. *P<0.05, **P<0.01 and ***P<0.001 indicate levels significantly different between naive and allergic mice. ATII-LCs = ATII-like cells; DCs = dendritic cells; Mϕ = macrophages.</p

Uptake of Bv1-Protein and Bv1-Peptide in A549 cells.

<p>The human ATII cell line A549 was incubated with 5 μg/ml of Bv1-Protein-FAM and Bv1-Peptide-FAM for 0.5, 1, 4, 24, and 48 hours. (<b>A</b>) The uptake was measured via flow cytometry. Dead cells were identified via 7-AAD staining and excluded from analysis. Data are the pool from three independently performed experiments of identical design. Values represent means ± SEM. A value P<0.05 was considered to be significant. *P<0.05, **P<0.01 and ***P<0.001 indicate levels significantly different between A549 cells stimulated with Bv1-Protein or Bv1-Peptide. (<b>B</b>) A549 cells were cultured for 4 hours with Bv1-Protein-FAM and Bv1-Peptide-FAM (green), transferred on glass slides, stained and mounted. Examination was performed with a confocal microscope. Nuclear counterstain was performed with DAPI (blue). ns = not significant.</p

Direct and transwell co-culture of A549 and THP1.

<p>A549 cells (5x10^<b>5</b> cells/well) were co-cultured with THP1 cells (5x10^<b>5</b> cell/well) directly or in a transwell system and stimulated with 5 μg/ml of Bv1-Protein and Bv1-Peptide or with 1 μg of LPS or 1 μg of Pam3-Cys for 6 days. IL-8 (<b>A</b>) and IL-6 (<b>B</b>) levels were measured in cell culture supernatants using ELISA. Values represent means ± SEM. A value P<0.05 was considered to be significant. **P<0.01 and ***P<0.001.</p