Developing a policy brief on physical activity promotion for children and adolescents

IntroductionWhile there are several approaches to collect basic information on physical activity (PA) promotion policies, some governments require more in-depth overviews on the situation in their country. In Germany, the Federal Ministry of Health expressed its interest in collecting detailed data on target group specific PA promotion, as relevant competences are distributed across a wide range of political levels and sectors. This study describes the development of a policy brief on physical activity promotion for children and adolescents in Germany. In particular, it addresses two major gaps in the current literature by systematically assessing good practice examples and “routine practices,” i.e., PA promotion activities already taking place on large scale and regular basis.Materials and methodsBased on relevant national and international guidelines, the TARGET:PA tool was co-produced by researchers and ministry officials. It includes (1) PA recommendations, (2) national prevalence rates, (3) recommendations for PA promotion, and data on national (4) routine practices, (5) good practice projects and (6) policies. Data were collected for children and adolescents in Germany using desk research, semi-structured interviews and secondary data analysis.ResultsA policy brief and scientific background document were developed. Results showed that 46% of the 4–5-year-olds fulfil WHO recommendations but only 15% of the 11–17-year-olds, and that girls are less active than boys. Currently, in Germany no valid data are available on the PA behaviour of children under the age of three. An overview of routine practices for PA promotion for children and adolescents was compiled, and experts were asked to critically assess their effectiveness, reach and durability. Overall, 339 target group specific projects for PA promotion were found, with 22 classified as examples of good practice. National PA policies for children and adolescents were identified across different sectors and settings.ConclusionThe study provides a comprehensive overview of the current status of PA promotion for children and adolescents in Germany. The co-production of the policy brief was a strength of the study, as it allowed researchers to take the needs of ministry officials into account, and as it supported the immediate uptake of results in the policymaking process. Future studies should test the applicability of the TARGET:PA tool to different target groups and countries

Triple Cytokine FluoroSpot Analysis of Human Antigen-Specific IFN-γ, IL-17A and IL-22 Responses

The involvement of T-helper (Th)1, Th17 and Th22 cell subsets, in immunity, as well as in pathological inflammatory reactions, makes it important to determine their relative proportion. A triple FluoroSpot detecting the hallmark cytokines of Th1 (IFN-γ), Th17 (IL-17A) and Th22 (IL-22) was developed and evaluated using human peripheral blood mononuclear cells from healthy donors incubated with tetanus toxoid, Candida albicans extract, mycobacterial purified protein derivative or medium only. Antigen stimulation yielded mainly cells secreting IFN-γ, IL-17A or IL-22 alone but lower proportions of double-secreting cells were also found; triple-secreting cells were rare. The response to C. albicans contrasted in that higher proportions of IL-17A single secreting as well as co-secreting cells, in particular IL-17A/IL-22, were found. The FluoroSpot analysis correlated well with single cytokine ELISpot assays ran in parallel and the methods displayed a comparable sensitivity. The results demonstrate the functionality of the FluoroSpot assay for simultaneous analysis of distinct Th1, Th17, Th22 as well as intermediate cell populations. The method provides a mean for a simple and rapid analysis of the involvement of these cells in immunity and disease

Consensus HIV-1 FSU-A integrase gene variants electroporated into mice induce polyfunctional antigen-specific CD4+ and CD8+ T cells

Our objective is to create gene immunogens targeted against drug-resistant HIV-1, focusing on HIV-1 enzymes as critical components in viral replication and drug resistance. Consensus-based gene vaccines are specifically fit for variable pathogens such as HIV-1 and have many advantages over viral genes and their expression-optimized variants. With this in mind, we designed the consensus integrase (IN) of the HIV-1 clade A strain predominant in the territory of the former Soviet Union and its inactivated derivative with and without mutations conferring resistance to elvitegravir. Humanized IN gene was synthesized; and inactivated derivatives (with 64D in the active site mutated to V) with and without elvitegravir-resistance mutations were generated by site-mutagenesis. Activity tests of IN variants expressed in E coli showed the consensus IN to be active, while both D64V-variants were devoid of specific activities. IN genes cloned in the DNA-immunization vector pVax1 (pVaxIN plasmids) were highly expressed in human and murine cell lines (>0.7 ng/cell). Injection of BALB/c mice with pVaxIN plasmids followed by electroporation generated potent IFN-γ and IL-2 responses registered in PBMC by day 15 and in splenocytes by day 23 after immunization. Multiparametric FACS demonstrated that CD8+ and CD4+ T cells of gene-immunized mice stimulated with IN-derived peptides secreted IFN-γ, IL-2, and TNF-α. The multi-cytokine responses of CD8+ and CD4+ T-cells correlated with the loss of in vivo activity of the luciferase reporter gene co-delivered with pVaxIN plasmids. This indicated the capacity of IN-specific CD4+ and CD8+ T-cells to clear IN/reporter co-expressing cells from the injection sites. Thus, the synthetic HIV-1 clade A integrase genes acted as potent immunogens generating polyfunctional Th1-type CD4+ and CD8+ T cells. Generation of such response is highly desirable for an effective HIV-1 vaccine as it offers a possibility to attack virus-infected cells via both MHC class I and II pathway

Comparison of Plasmid Vaccine Immunization Schedules Using Intradermal In Vivo Electroporation ▿

In vivo electroporation (EP) has proven to significantly increase plasmid transfection efficiency and to augment immune responses after immunization with plasmids. In this study, we attempted to establish an immunization protocol using intradermal (i.d.) EP. BALB/c mice were immunized with a plasmid encoding HIV-1 p37Gag, either i.d. with the Derma Vax EP device, intramuscularly (i.m.) without EP, or with combinations of both. A novel FluoroSpot assay was used to evaluate the vaccine-specific cellular immune responses. The study showed that i.d. EP immunizations induced stronger immune responses than i.m. immunizations using a larger amount of DNA and that repeated i.d. EP immunizations induced stronger immune responses than i.m. priming followed by i.d. EP boosting. Two and three i.d. EP immunizations induced immune responses of similar magnitude, and a short interval between immunizations was superior to a longer interval in terms of the magnitude of cellular immune responses. The FluoroSpot assay allowed for the quantification of vaccine-specific cells secreting either gamma interferon (IFN-γ), interleukin-2 (IL-2), or both, and the sensitivity of the assay was confirmed with IFN-γ and IL-2 enzyme-linked immunosorbent spot (ELISpot) assays. The data obtained in this study can aid in the design of vaccine protocols using i.d. EP, and the results emphasize the advantages of the FluoroSpot assay over traditional ELISpot assay and intracellular staining for the detection and quantification of bifunctional vaccine-specific immune responses

Average radiance at the sites of the IN/Luc-reporter genes co-injection correlates to IN-specific cytokine response.

<p>Inverse correlation of the bioluminescence represented by the average radiance (BLI) at the injection site on day 21 after the immunization to: % CD4+T cells secreting IFN-γ, IL-2, IFN-γ/IL-2, and IFN-γ/IL-2/TNF-α (<b>A</b>); % CD8+T cells secreting IFN-γ, TNF-α, IFN-γ/TNF-α and IFN-γ/IL-2/TNF-α (<b>B</b>). Correlations of BLI on days 4, 9, 15 and 21 to the triple IFN-γ/IL-2/TNF-α secretion by CD4+ T cells by day 23 (<b>C</b>). Results of the BLI and FACS analysis of the data collected in two independent experiments (2 times × 4 mice in each group) were analyzed by the Spearman rank-order test (Statistica AXA 10).</p

Integrase activities: 3′-processing and DNA strand transfer by IN variants.

<p>Products of 3′-processing and strand transfer of the synthetic DNA duplexes with ³²P-labeled B-strands (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone-0062720-t001" target="_blank">Table 1</a>) by the consensus HIV-1 clade A integrase (IN_a), its inactivated variant (IN_in), and the inactivated variant with elvitegravir resistance mutations (IN_in_e3) were separated by gel electrophoresis and quantified using Image-QuantTM 4.1 software. The 3′-processing assay: U5 substrate in the absence of integrases (lane 1) and in the presence of IN_a, IN_in, and IN_in_e3 (lanes 2, 3, and 4, respectively) (<b>A</b>). The strand transfer reaction: U5-2 substrate in the absence of integrases (lane 1) and in the presence of IN_a, IN_in, and IN_in_e3 (2, 3, and 4, respectively); T – the strand transfer products (<b>B</b>). Incubation of the non-specific DNA Ran in the absence of integrases (lane 1), and in the presence of IN_a, IN_in, and IN_in_e3 (2, 3, and 4, respectively) (<b>C</b>). Activities of HXB2 integrase, 3′-processing (1), strand transfer (2) (<b>D</b>). Tests were performed with 100 nM integrases and 10 nM DNA. Products were separated in denaturing 20% PAAG with 7M urea (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#s4" target="_blank">Methods</a> for details). Data are representative of two independent experiments.</p

Dynamics of bioluminescence at the sites of the IN gene and luciferase reporter gene co-administration.

<p><i>In vivo</i> monitoring of luciferase activity at days 4, 9, 15 and 21 after the administration of plasmids encoding the consensus IN (IN_a), inactivated consensus IN (IN_in), inactivated consensus IN with elvitegravir resistance mutations (IN_in_e3), or empty vector pVax1, each mixed with Luc reporter gene (1∶1). Images demonstrate two representative injection sites per group followed throughout the immunization. The scale to the right represents the strength of luminescent signal in pixels/sec/cmˆ2/sr (<b>A</b>). Kinetics of the luciferase expression over time (four mice in each group; two independent experiments) (<b>B</b>).</p

Peptides and peptide pools used in <i>in vitro</i> T-cell stimulation tests.

*<p>Choice of peptides done based on the epitopes mapped to these regions earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Rodriguez1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Casimiro1" target="_blank">[36]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Wilson1" target="_blank">[51]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Propato1" target="_blank">[52]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Sabbaj1" target="_blank">[53]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-Watanabe1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone.0062720-LubongSabado1" target="_blank">[95]</a>. Pool_CTL contains peptides representing human CTL epitopes mapped to the given regions.</p>**<p>IN series includes peptides recognized by human T cells; and MIN, by T cells of H2-K^d- restricted BALB/c mice.</p>***<p>Peptides of pool_CTL with mutations of resistance to elvitegravir where applicable.</p

IFN-γ/IL-2 Fluorospot assay of the splenocytes of mice immunized with IN gene variants.

<p>The results of IFN-γ/IL-2 Fluorospot performed on splenocytes of mice immunized with plasmids encoding consensus IN (IN_a), inactivated consensus IN (IN_in), inactivated consensus IN with mutations conferring resistance to elvitegravir (IN_in_e3), or empty vector. Splenocytes were stimulated <i>in vitro</i> with a Luc-derived peptide (LUC), and individual or pooled IN-derived peptides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#pone-0062720-t003" target="_blank">Table 3</a>) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062720#s4" target="_blank">Methods</a>. IN-specific <i>in vitro</i> secretion of IFN-γ (<b>A</b>), IL-2 (<b>B</b>), and dual secretion of IFN-γ/IL-2 (<b>C</b>). Responses represent the average number of signal-forming units (sfu) per mln cells in two independent experiment runs, each done in duplicate,+SD.</p