The Immune System – Multimedia

In order to infect a target cell, the HIV envelope glycoprotein gp120 has to interact with both the cellular CD4 receptor and a chemokine receptor, CCR5 or CXCR4, the so-called HIV co-receptors. Several flow cytometric/fluorometric assays are developed in our lab and are very helpful in deciphering the mode of action and interaction site of several classes of HIV entry inhibitors, such as the chemokine receptor antagonists.

The LacZ reporter fusion assay is an excellent method to precisely quantify knockdown efficiency of siRNA-sequences and can be corroborated by Western blot analysis. Efficient production of Lentiviruses and high transduction efficiency (up to 98%) were achieved on lymphoid B- and T-cells as demonstrated by FACS analysis and GFP expression. Successful knockdown effect with specific shRNA was observed in suspension cells three days after lentiviral infection.

Identification of low abundant proteins requires relative enrichment. We have tested systems for specific removal of high abundance proteins. Efficiency of albumin and IgG removal was assessed by ELISA and 2-DE. To estimate unspecific binding, six cytokines were spiked into serum. Recovery rates were determined with a cytometric bead assay.

The novel quantitative technique of "Targeted Profiling" of 1H 1D NMR data is applied to a unique model of rheumatoid arthritis. A number of candidate biomarkers not previously identified using NMR techniques are identified related to inflammation and immune response. The pharmaceutical relevance of several of these metabolites is discussed.

The Genarrayt 200+ Foods IgG test comprises of glass slides onto which 16 microarrays of over 200 different foods have been printed. Each microarray includes standards for quantitation and positive and negative controls for quality control. Food IgGs are detected by a novel fluorescent dye labelled anti-human IgG conjugate and results are measured using a laser scanner. Fluorescence intensity is directly proportional to antibody activity in the sample.

We have developed an autoantigen microarray that will allow laboratories to measure the presence of autoantibobies associated with different collagenois and vasculitis-related autoimmune diseases for a single serum sample.

Recent in vivo studies have demonstrated that transplanted MSC in mice were able to migrate into various tissues including lung, brain, bone marrow, heart and many others. Nevertheless, mechanisms responsible for the MSC migration remain unclear.

20 tuberculosis (12 slow-responders and 8 fast responders) patients were treated with directly observed short course anti-tuberculosis chemotherapy. Chest X-ray was performed. sICAM-1 and suPAR were measured in serum by ELISA, TNFRs using the luminex technology. General discrimination analysis on selected analytes gave, 91.66% and 87,50% correctly classify fast responders and slow responder respectively. The support vector machine analysis gave 100% correct classification.

Experimental approaches using short interfering RNA (siRNA) molecules to specifically silence gene expression have become more widely used in recent years. As for all techniques in molecular and cellular biology, the importance of protocol optimization and the use of appropriate controls cannot be overestimated.

Application of microarrays during process development helps to gain insight in biological processes involved in product formation, increasing process understanding.