Self-assembled Protein Arrays from an <i>Ornithodoros moubata</i> Salivary Gland Expression Library

Protein interactions play a critical role in the regulation of many biological events and their study in a high-throughput format has become a key area of proteomic research. Nucleid Acid Programmable Protein Arrays (NAPPA) technology allows the construction of protein arrays from cDNA expression libraries in high-throughput cell-free systems to study protein interaction and functions. Tick saliva contains antihemostatic, anti-inflammatory, and immunosuppressive proteins that counteract the host hemostatic, immune, and inflammatory responses allowing the ingestion of host blood and facilitating its infection by the tick-borne pathogens. Identification of such proteins and their functions could help in the selection of antigenic targets for the development of antitick and transmission-blocking vaccines. With that aim, we have prepared a cDNA expression library from the salivary glands of <i>Ornithodoros moubata</i> and subsequently produced a self-assembled protein microarray using 480 randomly selected clones from that library. The reproducibility of the array, its representativeness of the tick salivary protein repertoire, and the functionality of the in situ expressed proteins have been checked, demonstrating that it is a suitable tool for the identification and functional characterization of soft tick salivary molecules that interact with host proteins. Several clones in the array were shown to bind to human recombinant P-selectin. One of them was a likely secreted tick phospholipase A2, which may represent a potential new ligand for P-selectin. As these salivary molecules are likely involved in blood meal acquisition through the modulation of the host immune and hemostatic responses, this new high-throughput tool could open new avenues for development of new therapeutic agents and control strategies against ticks and tick-borne pathogens

Additional file 1: Table S1. of Ex vivo identification and characterization of a population of CD13high CD105+ CD45− mesenchymal stem cells in human bone marrow

List of antibodies used for the identification, characterization and isolation of bone marrow mesenchymal stem cells, including CD13high CD105+ and CD45– cells. (DOC 41 kb

Additional file 2: Figure S1. of Ex vivo identification and characterization of a population of CD13high CD105+ CD45− mesenchymal stem cells in human bone marrow

Representative bivariate dot plots and single parameter histograms showing the ex vivo expression profile of widely-accepted markers (CD271, MSCA-1, SSEA-4 and STRO-1) on bone marrow CD13high CD105+ CD45– cells. Panels A through C show the gating strategy used for the identification of these bone marrow cells based on their unique CD13high CD105+ CD45– features. Panels D through G illustrate the immunophenotypic features of bone marrow CD13high CD105+ CD45– cells from a representative healthy donor. Panel H summarizes the expression of CD271, MSCA-1, SSEA-4 and STRO-1 on CD13high CD105+ CD45– cells from five healthy donors. Baseline autofluorescence and expression levels for each protein are indicated in the histogram plot in gray and black, respectively, using the overlay histogram function of the Infinicyt software. On panel H the open circles represent individual samples, short horizontal and vertical lines display median values and the 95 % confidence interval of the levels of antigen expression (MFI values), respectively. SSC sideward light scatter, MFI median fluorescence intensity (arbitrary units scaled from 0 to 262,000). (TIFF 1440 kb

Proliferation index (percentage of S+G₂M cells) of different compartments of bone marrow (BM) cells in MDS patients grouped according to the World Health Organization (WHO) classification and the International Prognostic Scoring System (IPSS).

<p>Results expressed as median percentage of S+G₂ M cells and range between brackets. RA, refractory anemia; RCMD, refractory cytopenia with multilineage dysplasia; RAEB, RA with excess of blasts; MD/MPN, myelodysplastic/myeloproliferative neoplasms; INT, intermediate risk; NS, statistically not significantly different.</p>†<p>, p<0.05 and</p>††<p>, p<0.03 <i>vs.</i> normal/reactive BM.</p

Relationship between the degree of proliferation of non-lymphoid CD34⁺ and nucleated red blood cells from patients with myelodysplastic syndromes (MDS) and other haematological and biochemical characteristics of the disease.

<p>Results expressed as number of cases from all MDS patients analyzed and percentage between brackets.</p><p>PI, proliferation index;</p><p>LDH, lactate dehydrogenase;</p><p>AL, acute leukemia;</p><p>NS, statistically not significantly different.</p

Proliferation index (percentage of S+G₂M cells) of different BM cell compartments in normal/reactive BM (n = 94) and MDS patients (n = 106) with normal/favourable (n = 83) versus intermediate/poor (n = 23) cytogenetics.

<p>Results expressed as median percentage of cells and range between brackets.</p>*<p>, p<0.05 and</p>**<p>, p<0.03 <i>vs.</i> normal/reactive BM; and;</p>‡‡<p><b>,</b> p<0.03 <i>vs.</i> normal/favourable karyotype. Normal/favourable cytogenetics includes cases with a normal karyotype, -Y, del(5q) or isolated del(7q); poor cytogenetics includes cases with complex (≥3 chromosomal abnormalities) karyotypes and alterations of chromosome 7, except isolated del(7q), and; intermediate cytogenetics: other karyotypic abnormalities.</p

Proliferation index (percentage of S+G₂M cells) of different BM cell compartments in MDS <i>vs</i>. both AML and normal/reactive BM.

<p>Results expressed as median percentage of S+G₂/M cells and range between brackets. AML, acute myeloid leukemia; MDS, myelodysplastic syndrome; BM, bone marrow; NS, statistically not significantly different;</p>†<p>, MDS <i>vs</i>. normal/reactive BM;</p>¥<p>, MDS <i>vs</i>. AML;</p>*<p>, AML <i>vs.</i> normal/reactive BM.</p

Impact of currently used prognostic classifications and other disease features on overall survival and risk of transformation to acute leukemia (AL) of patients with myelodysplastic syndromes (MDS; n = 106).

<p>Overall survival (left column) and progression-free survival (transformation to AL; right column) curves are plotted for patients with MDS grouped according to the International Prognostic Scoring System (IPSS; row A), the World Health Organization-based Scoring System (WPSS; row B), the number of peripheral blood platelets at diagnosis (row C), the presence of multiple cytopenias (row D), transfusion dependency (row E), and serum LDH levels (row F).</p

Impact of the proliferation index (PI) of BM non-lymphoid (e.g.: myeloid plus immature) CD34⁺ precursors and nucleated red blood cells (NRBC) on the overall survival and progression (transformation to acute leukemia; AL) free survival of patients with myelodysplastic syndromes (MDS).

<p>Overall survival and progression-free survival curves are plotted for groups of MDS patients classified according to the PI of non-lymphoid (e.g.: myeloid plus immature) CD34⁺ cells (panels A to D) and NRBC (panels E to H). In the left column all MDS patients (n = 106) are analyzed together, while in the right column only MDS patients in the low plus intermediate-1 IPSS risk categories (n = 56) are considered.</p

Prognostic factors for overall survival (OS) and progression (transformation to acute leukemia)-free survival (PFS) in MDS.

<p>PI, proliferation index; NRBC, nucleated red blood cells; LDH, lactate dehydrogenase; AL, acute leukemia;</p>*<p>OS, overall survival expressed in months; PFS: progression to acute leukemia-free survival expressed in months; HR, hazard ratio.</p