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

Common Infectious Agents and Monoclonal B-Cell Lymphocytosis: A Cross-Sectional Epidemiological Study among Healthy Adults

<div><h3>Background</h3><p>Risk factors associated with monoclonal B-cell lymphocytosis (MBL), a potential precursor of chronic lymphocytic leukaemia (CLL), remain unknown.</p> <h3>Methods</h3><p>Using a cross-sectional study design, we investigated demographic, medical and behavioural risk factors associated with MBL. “Low-count” MBL (cases) were defined as individuals with very low median absolute count of clonal B-cells, identified from screening of healthy individuals and the remainder classified as controls. 452 individuals completed a questionnaire with their general practitioner, both blind to the MBL status of the subject. Odds ratios (OR) and 95% confidence interval (CI) for MBL were estimated by means of unconditional logistic regression adjusted for confounding factors.</p> <h3>Results</h3><p>MBL were detected in 72/452 subjects (16%). Increasing age was strongly associated with MBL (P-trend<0.001). MBL was significantly less common among individuals vaccinated against pneumococcal or influenza (OR 0.49, 95% confidence interval (CI): 0.25 to 0.95; P-value = 0.03 and OR: 0.52, 95% CI: 0.29 to 0.93, P-value = 0.03, respectively). Albeit based on small numbers, cases were more likely to report infectious diseases among their children, respiratory disease among their siblings and personal history of pneumonia and meningitis. No other distinguishing epidemiological features were identified except for family history of cancer and an inverse relationship with diabetes treatment. All associations described above were retained after restricting the analysis to CLL-like MBL.</p> <h3>Conclusion</h3><p>Overall, these findings suggest that exposure to infectious agents leading to serious clinical manifestations in the patient or its surroundings may trigger immune events leading to MBL. This exploratory study provides initial insights and directions for future research related to MBL, a potential precursor of chronic lymphocytic leukaemia. Further work is warranted to confirm these findings.</p> </div

Cytogenetic and molecular features of MBL^lo, MBL^hi and CLL B-cell clones.

<p>Results expressed as number of B-cell clones with cytogenetic abnormalities from all clones in the corresponding group (percentage) or as *median values of altered cells/clone (range). In 9 clones (1 MBL^lo,1MBL^hi and 7 CLL) biallelic del(13q14.3) was detected and hyperdiploidy was found in one MBL^lo clone. ^aMBL^hi vs CLL, ^bMBL^lo vs CLL, ^dMBL^lo plus MBL^hi vs CLL and ^eMBL^lo vs MBL^hi plus CLL. NS, no statistically significant differences observed (<i>P≥</i>0.05); CLL, chronic lymphocytic leukemia; MBL, monoclonal B-cell lymphocytosis.^†Includes the 5/66 cases with <i>NOTCH1</i> mutation associated to trisomy 12 in 3 cases, to biallelicdel(13q14.3) in one and to both monoallelic del(13q14.3) and del(17p) in the remaining cases.</p

Distribution of subjects included in the study and the corresponding CLL and CLL-like MBL clones, according to diagnosis.

*<p>For multiclonal CLL and CLL-like MBL cases as well as for other B-CLPD cases other than CLL, only CLL-like clones were considered; the later B-CLPD cases included the following diagnoses: HCL, hairy cell leukemia; SMZL/MALT, splenic marginal zone B-cell lymphoma/extranodal marginal zone B-cell lymphoma of mucosa-associated lymphoid tissue lymphoma. CLL, chronic lymphocytic leukemia; MBL, monoclonal B-cell lymphocytosis; B-CLPD, B-cell chronic lymphoproliferative disorders.</p>†<p>The number of clones per multiclonal case was of two in all diagnostic subgroups, except in three tri-clonal subjects corresponding to one CLL patient, one MBL^hi case and one patient with a B-CLPD other than CLL.</p

Principal component analysis (3-dimensionalX-Y-Z axis view of PC1 vs PC2 vs PC3, respectively) for comparison of “low-count MBL” (MBL^lo), “high-count MBL” (MBL^hi) and CLL B-cell clones according to the absolute number of clonal B cells/µL and the pattern of cytogenetic alterations (including the percentage of altered cells), using the Infinicyt^TMsoftware.

<p>Overall, MBL^lo, MBL^hi and CLL cases are clustered into groups distinguished by different colors in A: magenta, gray, and black circles (<b>A</b>). The distribution of MBL^lo, MBL^hi, CLL-stage A and CLL-stage B/C clones are coloured differently in B: MBL^lo, green; MBL^hi, red, CLL stage A and B/C light blue and dark blue, respectively (<b>B</b>). The most informative parameters contributing to the best discrimination between 1×1 comparisons of the three groups are displayed in a decreasing order of percentage contribution to each of the principal component (<b>C</b>); Distribution of MBL^lo, MBL^hi and CLL clones among the three major groups defined in panel A by principal component analysis (<b>D</b>); CLL, chronic lymphocytic leukemia; MBL, monoclonal B lymphocytosis; PC: principal component.</p

Odds ratios (OR) estimates, with 95% confidence intervals (CI), for “low-count” monoclonal B-cell lymphocytosis by self-reported family history of cancer.

<p>Ref: reference group; N: number; NA: not estimated; het: heterogeneity.</p><p>OR: Odds ratio; CI: confidence interval; 1^st degree relatives: parents, siblings and children; 2^nd degree relatives: grand-parents.</p>1<p>Adjusted for age (<50, 50–59, 60–69, 70+) and sex.</p>2<p>Further adjusted for family size (using the total number of children and siblings; categories: <6; 6 or more; missing).</p>3<p>Further adjusted for number of siblings (categories: <2; 3 or more; missing).</p>4<p>Further adjusted for number of children (<2; 3 or more; missing).</p>5<p>Adjusted for number of brothers (<3; 3 or more; missing).</p

Peripheral blood (PB) B-cell counts and BCR features of clonal MBL^lo, MBL^hi andCLL B cells.

<p>Results expressed as number of B-cell clones and percentage between brackets or as *median value (range). Statistically significant differences (<i>P</i><0.05) found between ^aMBL^hi vs CLL, ^bMBL^lo vs CLL, ^cMBL^lo vs MBL^hi, ^dMBL^lo plus MBL^hi vs CLL and ^eMBL^lo vs MBL^hi plus CLL. BCR, B-cell receptor; CLL, chronic lymphocytic leukemia; MBL, monoclonal B-cell lymphocytosis; SmIg, surface membrane immunoglobulin; <i>IGHV</i>, immunoglobulin heavy chain variable region genes.</p>†<p>Includes 6/117 cases with<5,000 clonal CLL B-cells/µL of PB, diagnosed with small lymphocytic lymphoma (SLL).</p

Odds ratios for “low-count” monoclonal B-cell lymphocytosis by selected potential variables related to infectious agents.

<p>Ref: reference group; N: number; het: heterogeneity; OR: Odds ratio; CI: confidence interval ¹: Adjusted for age (<50, 50–59, 60–69, 70+) and sex ²: Further adjusted for family size (using the total number of children and siblings; categories: <6; 6 or more; missing). ³: Further adjusted for number of siblings (categories: <2; 3 or more; missing) <i>⁴:</i> Further adjusted for number of children (0/2; 3or more; missing) <i>⁵:</i> Adjusted for age (<70, 70+) and sex, N_controls = 189; N_cases = 59 P-values were calculated using Wald-test. Black squares indicate OR, the area of each square being proportional to the amount of statistical information contributed. Horizontal lines represent 95% CI.</p

Prognostic Impact of del(17p) and del(22q) as Assessed by Interphase FISH in Sporadic Colorectal Carcinomas

<div><h3>Background</h3><p>Most sporadic colorectal cancer (sCRC) deaths are caused by metastatic dissemination of the primary tumor. New advances in genetic profiling of sCRC suggest that the primary tumor may contain a cell population with metastatic potential. Here we compare the cytogenetic profile of primary tumors from liver metastatic versus non-metastatic sCRC.</p> <h3>Methodology/Principal Findings</h3><p>We prospectively analyzed the frequency of numerical/structural abnormalities of chromosomes 1, 7, 8, 13, 14, 17, 18, 20, and 22 by iFISH in 58 sCRC patients: thirty-one non-metastatic (54%) <em>vs.</em> 27 metastatic (46%) disease. From a total of 18 probes, significant differences emerged only for the 17p11.2 and 22q11.2 chromosomal regions. Patients with liver metastatic sCRC showed an increased frequency of del(17p11.2) (10% <em>vs.</em> 67%;p<.001) and del(22q11.2) (0% <em>vs.</em> 22%;p = .02) versusnon-metastatic cases. Multivariate analysis of prognostic factors for overall survival (OS) showed that the only clinical and cytogenetic parameters that had an independent adverse impact on patient outcome were the presence of del(17p) with a 17p11.2 breakpoint and del(22q11.2). Based on these two cytogenetic variables, patients were classified into three groups: low- (no adverse features), intermediate- (one adverse feature) and high-risk (two adverse features)- with significantly different OS rates at 5-years (p<.001): 92%, 53% and 0%, respectively.</p> <h3>Conclusions/Significance</h3><p>Our results unravel the potential implication of del(17p11.2) in sCRC patients with liver metastasis as this cytogenetic alteration appears to be intrinsically related to an increased metastatic potential and a poor outcome, providing additional prognostic information to that associated with other cytogenetic alterations such as del(22q11.2). Additional prospective studies in larger series of patients would be required to confirm the clinical utility of the new prognostic markers identified.</p> </div

Clinical characteristics of GBM patients (n = 46) included in this study with information about the type of SNP-arrays used in each case to investigate DNA CN alterations.

<p>Surgical removal: ST- subtotal; T- total.</p>*<p>Tumors analyzed by gene expression arrays.</p>#<p>Only patient that remained alive at the moment of closing the study; all other patients had died.</p