Label-Free Quantitative Proteomics Distinguishes General and Site-Specific Host Responses to Pseudomonas aeruginosa Infection at the Ocular Surface.

Mass spectrometry-based proteomics enables the unbiased and sensitive profiling of cellular proteomes and extracellular environments. Recent technological and bioinformatic advances permit identifying dual biological systems in a single experiment, supporting investigation of infection from both the host and pathogen perspectives. At the ocular surface, Pseudomonas aeruginosa is commonly associated with biofilm formation and inflammation of the ocular tissues, causing damage to the eye. The interaction between P. aeruginosa and the immune system at the site of infection describes limitations in clearance of infection and enhanced pathogenesis. Here, the extracellular environment (eye wash) of murine ocular surfaces infected with a clinical isolate of P. aeruginosa is profiled and neutrophil marker proteins are detected, indicating neutrophil recruitment to the site of infection. The first potential diagnostic markers of P. aeruginosa-associated keratitis are also identified. In addition, the deepest murine corneal proteome to date is defined and proteins, categories, and networks critical to the host response are detected. Moreover, the first identification of bacterial proteins attached to the ocular surface is reported. The findings are validated through in silico comparisons and enzymatic profiling. Overall, the work provides comprehensive profiling of the host-pathogen interface and uncovers differences between general and site-specific host responses to infection.

Establishment a CHO Cell Line Expressing Human CD52 Molecule.

BACKGROUND: CD52 is a small glycoprotein with a GPI anchor at its C-terminus. CD52 is expressed by Normal and malignant T and B lymphocytes and monocytes. There are detectable amounts of soluble CD52 in plasma of patients with CLL and could be used as a tumor marker. Although the biological function of CD52 is unknown but it seems that CD52 may be involved in migration and activation of T-cells .The aim of this study was to clone and express human CD52 gene in CHO cell line and studying its function in more details. METHODS: Based on GenBank databases two specific primers were designed for amplification of cd52 gene. Total RNA was extracted from Raji cell line and cDNA synthesized. Amplified fragment was cloned in pBudCE4.1 vector. The new construct was transfected to CHO-K1 cell line using electroporation method. Expression of recombinant CD52 protein was evaluated by Real time PCR and flow cytometry methods. RESULTS: Amplification of CD52 gene using specific primers on Raji cDNA showed a 209 bp band. New construct was confirmed by PCR and restriction pattern and sequence analysis. The new construct was designated as pBudKT1. RT-PCR analysis detected cd52 mRNAs in transfected cells and Flow cytometry Results showed that 78.4 % of cells represented CD52 in their surfaces. CONCLUSION: In conclusion, we established a human CD52 positive cell line, CHO-CD52, and the protein was expressed on the membrane. Cloning of the CD52 gene could be the first step for the production of therapeutic monoclonal antibodies and detection systems for soluble CD52 in biological fluids.