Volunteers
The experiment was approved by the local ethics committee (registration number 708/98). After medical examination, 15 healthy male volunteers under the age of 30 were enrolled in this phase one clinical study. Each volunteer was exposed to a single dose of the contrast agent LK565 applied intravenously. Four participants were exposed to the contrast agent LK565 for the second time because they had already been involved in a prior study [11]. The 15 volunteers were divided up into 3 groups (n = 5). The first group received 0.15 mg/kg bodyweight, the second group 0.4 mg/kg bodyweight and finally the third group 0.7 mg/kg LK565.
Contrast agent
Prior to application, the contrast agent LK565 was dissolved in 10 ml physiological sodium chloride solution (37°C) under sterile conditions. To eliminate aggregations, the solution was filtered using a 20 μm mesh just before intravenous injection.
Sampling
Heparinized blood samples (2 ml) were taken before application (t = 0), after 2 h, 6 h, 24 h, 48 h, 72 h, 96 h, and 1 ml after 10 d, 12 d and 14 d. For routine analysis, additional blood samples were drawn before and 2 hours after intravenous contrast agent injection. The volunteers were under medical care throughout the whole experiment.
Phagocytosis
Phagocytosis capacity was determined in vitro before (t = 0) and after 2 h, 6 h and 24 h with Phagotest™ (Becton Dickinson, Germany) and analyzed via flow cytometry (FACScalibur™, BectonDickinson, Germany). FITC-labeled E. coli, which were opsonized with antibodies and complement factors, were incubated with leukocytes. After incubation, the increased fluorescence of the phagocytes was due to the uptake of bacteria. The fluorescence intensity is dependent on the amount of phagocytosed bacteria. A sample chilled on ice was used for control.
Tumor necrosis factor alpha (TNF-α)
Intracellular TNF-α production in monocytes and macrophages was analyzed before (t = 0) and after 2 h, 6 h, and 24 h. For detection, an anti-human TNF-α Pycoerythrin (PE) monoclonal antibody (mAb) was applied (Pharmingen, Germany). The specificity of the antibody had been validated in blocking experiments. To 40 μl blood, 145 μl RPMI and 15 μl 40 μM Monensin were added (Monensin prevents cytokine release by blocking the golgi apparatus). Cells were incubated for 2 h at T = 37°C and 5% CO2 atmosphere. After washing, monocytes were surface stained by anti-human CD14 TriColor mAb (Medac, Germany). Red blood cells were lysed using FACS™ Lysing Solution (Becton Dickinson, Germany). Cells were fixed and permeabilized by addition of 250 μl Cytofix/Cytoperm™ (Becton Dickinson, Germany). Finally, the retained intracellular TNF-α was stained by the anti-TNF-α PE mAb. For a positive control, cells were stimulated with 0.25 μg Lipopolysaccharide (LPS), following the same procedure as mentioned above.
Surface activation markers
Surface activation markers were analyzed before (t = 0) and after 2 h, 6 h, 24 h, 48 h, 72 h, and 96 h. For the detection of activation markers, anti-human CD69 PE (Holzel Diagnostika, Germany), CD25 Fluoresceinisothiocyanat (FITC), CD71 FITC, CD11b PE (Diaclone Research, Germany) and HLA-DR PerCP (Becton Dickinson, Germany) mAbs were used. For a positive control, cells were stimulated with 10 ng/ml Phorbol-12-Myristat-13-Acetat (PMA) and 1 μg/ml Ionomycin for 2 h, while macrophages/monocytes were stimulated with 1.25 μg/ml LPS for 2 h. To distinguish between leukocytes, additional surface markers, anti-human CD45 Allophycocyanine (APC), CD19 APC (Caltag Laboratories, Germany), CD3 Tri/Color (DAKO, Germany) and CD14 FITC (Diaclone Research, Germany) mAbs were used for detection. Blood samples (40 μl) were stained for 4-color flow cytometry analysis. After staining, red blood cells were lysed, subsequently leukocytes were analyzed. Antibody specificity was validated by isotype controls (Becton Dickinson, Germany).
Specific antibody development
Serum samples were analyzed before (t = 0) and after 24 h, 48 h, 72 h, 96 h, 10 d, 12 d, and 14 d. Specific anti-LK565 antibodies were determined via indirect ELISA. Indirect ELISA was performed in a 96-well plate by LK565 immobilization overnight. Optimal concentrations had previously been determined by common cross-testing. Serum was obtained from blood samples after centrifugation at 400 G. After incubation for 2 h at room temperature, detector antibodies (Biozol Diagnostica GmbH, Germany) were added and incubated at 4°C overnight. Washing was carried out with phosphate buffered saline (PBS) and blocking buffer. For detection, alkaline phosphatase (AP)-linked goat-anti-human IgG and IgM antibodies with para-Nitrophenylphosphate (p-NPP) as substrate (Biozol Diagnostica GmbH, Germany) were used. After development, the enzyme reaction was stopped with 0.5 M NaOH and measured at λ = 405 nm. For the functional (positive) control of the indirect ELISA, total immunoglobuline was captured polyclonally. For a negative control, the serum of a person who had not been exposed to the contrast agent was obtained.