The trypanocidal activity of photoreactive azido analogs of ethidium was tested to determine the suitability of using such compounds as in vivo probes to study the mechanism of the antitrypanosomal activity of ethidium. Eight ethidium analogs, including three nonphotoreactive compounds, were tested for their ability to kill T. brucei both with and without photolytic activation. Two analogs tested, the monoamino-monoazido isomers, showed greater that 100-fold enhancement of trypanocidal activity following photolytic activation in situ. Without photolytic activation, only the nonphotoreactive monoamino precursor analogs showed activity greater than the parent ethidium compound. The availability of suitable ethidium analogs which can be covalently attached by in situ photoactivation provides a new approach for studying the mechanism by which ethidium exerts its trypanocidal activity.
Human Interferon-gamma (IFN-y) ELISpot assays are designed for the detection of IFN-y secreting cells at the single cell level. We have developed and validated an ELISpot assay with enhanced sensitivity (SC) and compared its performance relative to a common commercial kit (CK). Three manufactured lots of ELISpot kits (SC1-3) and one CK were analyzed in cellular titration experiments using PHA stimulation of cryopreserved PBMC from four subjects. The minimum quantity of cells required to elicit a single spot-forming cell (SFC) per well was 25,000 for CK compared to 7,000 for SC. In many cases, > 50,000 cells were required to measure a response using the CK. Slopes of log/log plots were 2.3 for CK versus 1.7 for SC, with no overlap at the 95% CI level. At the upper detection limit (200,000 cells/well), the CK averaged 108±46 SFC/well, while SC 1-3 measured 264±79, 377±120 and 231 ±95 SFC/well, respectively, demonstrating a broader linear range. Differences in sensitivity and range are possibly due to spot size, SC produced spots 9 x 10-3 mm2 (95% CI 7-11) while CK averaged 19 x 10-3 mm2 (95% CI 17-21), P < 0.001. The same PBMC were also analyzed using CEF (CMV, EBV and Influenza A – specific peptide pools) and CMV peptide stimulants and media controls. For CEF, the mean values for SC 1-3 were 100.0, 90.0, and 86.6 SFC/well, vs. 77.7 for CK. The CMV responses were 104.3, 80.2 and 74.4 SFC/well for SC 1-3 vs. 77.1 for CK. PBMC added with no stimulus, gave backgrounds of <2 SFC/well in all cases. The average within run coefficients of variation (CV) were similar- 10.2, 14.1 and 18.9 for SC 1-3 and 13.8 for CK when stimulated using CEF and 20.8 to 26.4 with CMV. No significant differences were observed for means or variability between CK and SC 1-3. In summary, SeraCare has developed and validated an IFN-y ELISpot kit having an enhanced dynamic range and sensitivity.
Cryopreservation (CP) of peripheral blood mononuclear cells (PBMC) to remain functional upon thawing is important for their use in immunotherapy studies. Sera are often used for CP, but demonstrate variability and are replete with growth factors that can result in high backgrounds during cytokine assays. We compared two human serum albumins (HSA) [Grifols® (G) and Bayer® (B)] in the formulation of PBMC CP media containing DMSO (CryoMedia™), and monitored post‐thaw recovery, viability, and secretion of interferon‐γ (IFN‐γ) by ELISpot in response to PHA, CMV and CEF peptide pool stimuli. PBMCs from a leukopack were washed then cryopreserved at 1.5×107/ml; viability prior to CP was approx. 98%. After storage in LN2 vapor mean recoveries were 80.4 (95% CI 55.6–105.2) and 68.0 (95% CI 64.4–71.6) for G and B, respectively, (p=0.4). Post‐thaw viabilities were 92% and decreased respectively to 74 % and 79% for G and B after 48 hours. In response to PHA stimuli, mean IFN‐γ ELISpot response was 1024 (95% CI 996–1051) and 436 (95% CI 407–46) SFC/well using PBMC formulated in B and G HSA, respectively (p < 0.001). Differences were also observed in the response to CMV peptide stimuli using B [18.4 (95% CI 15.7–21.2)] and G [15.4 (95% CI 13.3–17.5)], p < 0.05. Response to CEF peptides for both formulations was similar [16.6 (95% CI 13.3–20.0), B] and [17.4 (95% CI 14.7–20.1), G], p = 0.7. Background to media containing no stimuli were <1 SFC/well for both. The data suggest differences in HSA formulations used during CP of PBMC may elicit significantly different cellular immune responses to select peptide antigens. Additional PBMC donors and studies of proliferation and apoptosis are in process to further elucidate the functional effects of specific manufactures’ HSA during PBMC CP.
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