Comparison of different label-free imaging high-throughput biosensing systems for aptamer binding measurements using thrombin aptamers.

To enable the analysis of several hundreds to thousands of interactions in parallel, high-throughput systems were developed. We used established thrombin aptamer assays to compare three such high-throughput imaging systems as well as analysis software and user influence. In addition to our own iRIf-system, we applied bscreen and IBIS-MX96. As non-imaging reference systems we used Octet-RED96, Biacore3000, and Monolith-NT.115. In this study we measured 1378 data points. Our results show that all systems are suitable for analyzing binding kinetics, but the kinetic constants as well as the ranking of the selected aptamers depend significantly on the applied system and user. We provide an insight into the signal generation principles, the systems and the results generated for thrombin aptamers. It should contribute to the awareness that binding constants cannot be determined as easily as other constants. Since many parameters like surface chemistry, biosensor type and buffer composition may change binding behavior, the experimenter should be aware that a system and assay dependent KD is determined. Frequently, certain conditions that are best suited for a given biosensing system cannot be transferred to other systems. Therefore, we strongly recommend using at least two different systems in parallel to achieve meaningful results.

Comparison of two methods for enumerating circulating tumor cells in carcinoma patients.

BACKGROUND: Monitoring of circulating tumor cells (CTCs) in blood of carcinoma patients treated with novel compounds may be a measurement of treatment effectiveness. Before it can be used clinically, a reliably method is needed to enumerate CTCs. We compared two methods for CTC enumeration, OnkoQuick and the CellSearch system. METHODS: We drew 22.5 ml of blood into three CellSave tubes from 15 healthy donors and 61 patients with metastatic carcinoma. After pooling, 15 ml was processed with OncoQuick and 7.5 ml with CellSearch. RESULTS: With both methods no CTCs were found in healthy donors. At least one CTC was detected in 14 of 61 patients (23%) with OncoQuick and 33 of 61 patients (54%) with CellSearch (P < 0.0001). The number of CTCs detected was larger for CellSearch (mean 20 CTCs/7.5 ml of blood) than for OncoQuick (3 CTCs/7.5 ml; P < 0.0001). CONCLUSION: The CellSearch system is a more accurate and sensitive method to enumerate CTCs. Further studies are warranted to evaluate CTC enumeration by the CellSearch system as a monitoring tool for the evaluation of the efficacy of novel anticancer agents.

Vgamma9Vdelta2 T cells recovered from eyes of patients with Behçet's disease recognize non-peptide prenyl pyrophosphate antigens.

The phenotype and antigen-specificity of T cells expanded by mitogenic stimulation from intra-ocular fluid (IOF) samples of affected eyes of six Behçet's disease (BD) patients, and seven patients with other uveitis entities, were determined. High numbers of gammadelta T cells, predominantly Vgamma9Vdelta2 T cells, were only detected in the IOF-derived TCL of three BD patients. Whereas no TCL responded to heat shock protein (HSP) 65 kDa, reactivity to isopentyl pyrophosphate (IPP) and related non-peptide prenyl pyrophosphates (PPP) was restricted to the gammadelta T cell containing TCL. Upon IPP stimulation, these TCL secreted IFN-gamma but no IL-4. By single-cell analysis of intracellular IFN-gamma production and CD69 expression the IOF-derived IPP-specific T cells were identified as CD4(-)CD8(-) gammadelta T cells. The data presented suggest the infiltration of PPP-specific Vgamma9Vdelta2 Th1-like cells into the eye of BD patients with uveitis.

Restricted T cell receptor beta-chain variable region protein use by cornea-derived CD4+ and CD8+ herpes simplex virus-specific T cells in patients with herpetic stromal keratitis.

Herpetic stromal keratitis (HSK) is a T helper type 1 cell-mediated inflammatory disease triggered by herpes simplex virus (HSV) infection of the cornea. In contrast to animal models of HSK, little is known about the role of T cells in human HSK. The phenotypes and repertoires of HSV-specific T cells recovered from the corneas of 12 patients with HSK were determined by flow cytometry. Cornea-derived T cell lines (TCLs) from 10 of the 12 patients contained high numbers of HSV-specific T cells. HSV reactivity was HSV type common and involved relatively more CD8(+) than CD4(+) T cells. The majority of the TCLs showed restricted T cell receptor beta-chain variable region protein (TCRBV) use. T cells expressing 1 or 2 TCRBVs dominated the HSV-1 reactivity in 3 of 5 TCLs analyzed. The data demonstrate that both CD4(+) and CD8(+) T cells may be involved in the HSV-specific T cell response in the corneas of patients with HSK and suggest that restricted TCRBV use by cornea-residing HSV-specific T cells occurs.