Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-μl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 μl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-μl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

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With the development of online data collection and instruments such as Amazon's Mechanical Turk (MTurk), the appearance of malicious software that generates responses to surveys in order to earn money represents a major issue, for both economic and scientific reasons. Indeed, even if paying one respondent to complete one questionnaire represents a very small cost, the multiplication of botnets providing invalid response sets may ultimately reduce study validity while increasing research costs. Several techniques have been proposed thus far to detect problematic human response sets, but little research has been undertaken to test the extent to which they actually detect nonhuman response sets. Thus, we proposed to conduct an empirical comparison of these indices. Assuming that most botnet programs are based on random uniform distributions of responses, we present and compare seven indices in this study to detect nonhuman response sets. A sample of 1,967 human respondents was mixed with different percentages (i.e., from 5% to 50%) of simulated random response sets. Three of the seven indices (i.e., response coherence, Mahalanobis distance, and person-total correlation) appear to be the best estimators for detecting nonhuman response sets. Given that two of those indices-Mahalanobis distance and person-total correlation-are calculated easily, every researcher working with online questionnaires could use them to screen for the presence of such invalid data.

We have recently shown that mice with a targeted disruption of CCR2, the receptor for monocyte chemoattractant protein-1, have markedly impaired recruitment of macrophages to sites of inflammation. An unexpected finding in the CCR2−/− mice was a dramatic decrease in the production of IFN-γ after challenge with purified protein derivative of Mycobacterium bovis. In this study, we have investigated the mechanism of this cytokine production defect. In vitro, direct activation of splenocytes with CD3/CD28 Abs failed to reveal any differences in IFN-γ production between CCR2+/+ and CCR2−/− mice. However, after immunization, the number of Ag-specific, IFN-γ-producing cells in the draining lymph nodes was decreased by 70% in the CCR2−/− mice, suggesting an in vivo trafficking defect. Direct measurement of cell trafficking with fluorescently labeled CFA revealed a marked decrease in the number of monocytes/macrophages migrating to the site of immunization and to the draining lymph nodes in the CCR2−/− mice. The data suggest that impaired trafficking of APCs in the CCR2−/− mice contributes to the defect in IFN-γ production. These data support the idea that CCR2-positive monocytes/macrophages are critical in linking the innate and adaptive immune responses.