Proceedings of the 3rd Biennial Conference of the Society for Implementation Research Collaboration (SIRC) 2015: advancing efficient methodologies through community partnerships and team science

It is well documented that the majority of adults, children and families in need of evidence-based behavioral health interventionsi do not receive them [1, 2] and that few robust empirically supported methods for implementing evidence-based practices (EBPs) exist. The Society for Implementation Research Collaboration (SIRC) represents a burgeoning effort to advance the innovation and rigor of implementation research and is uniquely focused on bringing together researchers and stakeholders committed to evaluating the implementation of complex evidence-based behavioral health interventions. Through its diverse activities and membership, SIRC aims to foster the promise of implementation research to better serve the behavioral health needs of the population by identifying rigorous, relevant, and efficient strategies that successfully transfer scientific evidence to clinical knowledge for use in real world settings [3]. SIRC began as a National Institute of Mental Health (NIMH)-funded conference series in 2010 (previously titled the “Seattle Implementation Research Conference”; $150,000 USD for 3 conferences in 2011, 2013, and 2015) with the recognition that there were multiple researchers and stakeholdersi working in parallel on innovative implementation science projects in behavioral health, but that formal channels for communicating and collaborating with one another were relatively unavailable. There was a significant need for a forum within which implementation researchers and stakeholders could learn from one another, refine approaches to science and practice, and develop an implementation research agenda using common measures, methods, and research principles to improve both the frequency and quality with which behavioral health treatment implementation is evaluated. SIRC’s membership growth is a testament to this identified need with more than 1000 members from 2011 to the present.ii SIRC’s primary objectives are to: (1) foster communication and collaboration across diverse groups, including implementation researchers, intermediariesi, as well as community stakeholders (SIRC uses the term “EBP champions” for these groups) – and to do so across multiple career levels (e.g., students, early career faculty, established investigators); and (2) enhance and disseminate rigorous measures and methodologies for implementing EBPs and evaluating EBP implementation efforts. These objectives are well aligned with Glasgow and colleagues’ [4] five core tenets deemed critical for advancing implementation science: collaboration, efficiency and speed, rigor and relevance, improved capacity, and cumulative knowledge. SIRC advances these objectives and tenets through in-person conferences, which bring together multidisciplinary implementation researchers and those implementing evidence-based behavioral health interventions in the community to share their work and create professional connections and collaborations

A Novel Mouse Model of <i>Campylobacter jejuni</i> Gastroenteritis Reveals Key Pro-inflammatory and Tissue Protective Roles for Toll-like Receptor Signaling during Infection

<div><p><i>Campylobacter jejuni</i> is a major source of foodborne illness in the developed world, and a common cause of clinical gastroenteritis. Exactly how <i>C. jejuni</i> colonizes its host's intestines and causes disease is poorly understood. Although it causes severe diarrhea and gastroenteritis in humans, <i>C. jejuni</i> typically dwells as a commensal microbe within the intestines of most animals, including birds, where its colonization is asymptomatic. Pretreatment of C57BL/6 mice with the antibiotic vancomycin facilitated intestinal <i>C. jejuni</i> colonization, albeit with minimal pathology. In contrast, vancomycin pretreatment of mice deficient in SIGIRR (<i>Sigirr^−/−</i>), a negative regulator of MyD88-dependent signaling led to heavy and widespread <i>C. jejuni</i> colonization, accompanied by severe gastroenteritis involving strongly elevated transcription of Th1/Th17 cytokines. <i>C. jejuni</i> heavily colonized the cecal and colonic crypts of <i>Sigirr^−/−</i> mice, adhering to, as well as invading intestinal epithelial cells. This infectivity was dependent on established <i>C. jejuni</i> pathogenicity factors, capsular polysaccharides (<i>kpsM</i>) and motility/flagella (<i>flaA</i>). We also explored the basis for the inflammatory response elicited by <i>C. jejuni</i> in <i>Sigirr^−/−</i> mice, focusing on the roles played by Toll-like receptors (TLR) 2 and 4, as these innate receptors were strongly stimulated by <i>C. jejuni</i>. Despite heavy colonization, <i>Tlr4^−/−/Sigirr^−/−</i> mice were largely unresponsive to infection by <i>C. jejuni</i>, whereas <i>Tlr2^−/−/Sigirr^−/−</i> mice developed exaggerated inflammation and pathology. This indicates that TLR4 signaling underlies the majority of the enteritis seen in this model, whereas TLR2 signaling had a protective role, acting to promote mucosal integrity. Furthermore, we found that loss of the <i>C. jejuni</i> capsule led to increased TLR4 activation and exaggerated inflammation and gastroenteritis. Together, these results validate the use of <i>Sigirr^−/−</i> mice as an exciting and relevant animal model for studying the pathogenesis and innate immune responses to <i>C. jejuni</i>.</p></div

TLR2 and 4 reporter assays.

<p>HEK-Blue hTLR2 (A) and HEK-Blue hTLR4 (B) reporter cell lines were exposed for 4 hrs to either live and heat-killed wildtype <i>C. jejuni</i> 81–176, <i>ΔkpsM</i> or <i>ΔkpsM+kpsM</i>. The wild-type <i>C. jejuni</i> stimulates both TLR2 and TLR4 in a dose-dependent fashion. The <i>ΔkpsM</i> mutant significantly increased the signaling by both TLR2 and TLR4, as indicated by the assay, with the increase in stimulation also being in a dose-dependent manner, except for the TLR4 assay where the readers were near the maximum for both the 20 and 200 MOI readings. The complemented <i>ΔkpsM+kpsM</i> strain completely restored the wild-type phenotype with TLR2 and mostly restored the phenotype with TLR4. Values represent the mean of three independent experiments and statistical significance was determined by a two-way ANOVA with a Bonferroni post-test. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

Colonization and pathology of <i>Sigirr^−/−</i> and TLR-deficient mice by <i>C. jejuni ΔkpsM in vivo</i>.

<p>(A) H&E stained histological sections of ceca recovered from <i>Sigirr^−/−</i>, <i>Tlr2^−/−/Sigirr^−/−</i> and <i>Tlr4^−/−/Sigirr^−/−</i> mice, colonized with <i>C. jejuni ΔkpsM</i> 7 DPI, at 100× magnification. Very severe inflammation is evident in the <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice, however once again, no significant pathology was evident in the <i>Tlr4^−/−/Sigirr^−/−</i> mice. (B) Immunofluorescence of <i>Sigirr^−/−</i> mice infected by <i>C. jejuni ΔkpsM</i>, 7 DPI. Sections are stained for DAPI (blue), β-actin (green), and <i>C. jejuni</i> (red). <i>Sigirr^−/−</i> mice exhibit significant neutrophil infiltration, hyperplasia, and <i>C. jejuni ΔkpsM</i> is clearly visible in large masses within the cecal crypts. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. Only <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice at 7 DPI showed a significant increase in pathology (****p<0.0001), relative to the uninfected <i>Sigirr^−/−</i> control. The <i>Tlr2^−/−/Sigirr^−/−</i> mice also exhibited statistically significantly higher inflammation at 7 DPI relative to <i>Sigirr^−/−</i> mice also at 7 DPI (**p<0.001). In contrast, none of the mouse strains at 3 DPI showed any statistically significant increase in pathology, relative to control mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test.</p

Immunofluorescent staining of intracellular <i>C. jejuni in vivo</i>.

<p>(A) Intracellular <i>C. jejuni</i> are visible in the cecal epithelium. <i>C. jejuni</i> (red), are visible against the β-actin (green) and the nuclei (DAPI, blue) of the cecal epithelium of a <i>Sigirr^−/−</i> mouse, ×1000 magnification. (B) Confocal image of <i>C. jejuni</i> (red) present within epithelial cells of the colon of a <i>Sigirr^−/−</i> mouse, highlighted against the Cytokeratin 19 of the cytoskeleton (green) and the nuclei (blue), with the z-stack cross-section indicating the <i>C. jejuni</i> within the cell. (C) Cross-section of a Z-stack, of a colonic epithelial cell of a <i>Sigirr^−/−</i> mouse. The internalized <i>C. jejuni</i> (red) are clearly visible within the cytoplasm of the cell, as outlined by the β-actin (green) along the edge of the cell. (D) Internalized <i>C. jejuni</i> (red) co-localize with LAMP-1 positive (green) vesicles present within epithelial cells of a <i>Sigirr^−/−</i> mouse colon.</p

Colonization of WT and <i>Sigirr−/−</i> mice by <i>ΔkpsM</i> and <i>ΔflaA</i>.

<p>Colonization of WT and <i>Sigirr^−/−</i> mice by <i>ΔkpsM</i> (A) and <i>ΔflaA</i> (B) and their respective complemented strains (<i>ΔkpsM</i>+<i>kpsM</i> and <i>ΔflaA</i>+<i>flaA</i>), at both 3 and 7 DPI. The <i>ΔkpsM</i> mutant exhibited reduced colonization at 3 DPI only, while the <i>ΔflaA</i> mutant was unable to colonize at either 3 or 7 DPI. The complemented <i>ΔflaA+flaA</i> colonized at high numbers, similar to wild-type. Statistical significance was determined by a Mann-Whitney test, ***p<0.001. n = 7–10 mice for the <i>ΔkpsM</i> mutant and complement, n = 5–7 mice for the <i>ΔflaA</i> mutant and complement. (C) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i> at ×100 magnification. No noticeable inflammation was evident in either WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔflaA</i>. (D) H&E stained histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice infected with <i>C. jejuni ΔkpsM</i> 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. WT mice did not exhibit signs of inflammation when infected with <i>C. jejuni ΔkpsM</i>, however <i>Sigirr^−/−</i> mice exhibited signs of severe inflammation at 7 DPI.</p

Cytokine production in infected mice.

<p>(A–H) RT-qPCR conducted on RNA extracted from the ceca of uninfected control or infected mice. Controls are the pooled results of 9, vancomycin pre-treated, but uninfected mice, euthanized 3 days post-treatment. All infected mice represent the average results of 3 independent experiments, each of which include the pooled RNA of 2–3 mice, for 6–9 mice total for each mouse strain, euthanized either 3 or 7 DPI. Statistical significance was determined using a One way ANOVA with a Bonferroni post-test. * p<0.05 relative to WT (B6) or <i>Sigirr^−/−</i> uninfected control mice. ** p<0.05 relative to the infected WT (B6) mice euthanized on the same DPI in addition to the uninfected control mice.</p

Colonization of WT and <i>Sigirr−/−</i> mice by <i>C. jejuni</i> 81–176, 3 and 7 DPI.

<p>(A) High numbers (∼10⁹ CFUs/g) of <i>C. jejuni</i> were recovered at both 3 and 7 DPI from the ceca of infected mice that were pre-treated with 5 mg of vancomycin. No statistically significant differences in numbers were found between WT and <i>Sigirr^−/−</i> mice as indicated by a t-test, p>0.05. n = 10 or 11 WT mice, and 12 or 13 <i>Sigirr^−/−</i> mice for 3 and 7 DPI respectively. (B) H&E stained, formalin-fixed histological sections of ceca recovered from WT or <i>Sigirr^−/−</i> mice 3 and 7 DPI. Upper panels are ×100 magnification, while lower panels are ×400 magnification. (C) Pathological scoring was done by two blinded observers, using H&E stained, formalin-fixed cecal tissue sections. Each condition represents a minimum of three separate experimental replicates, with 2–3 mice per experiment for a total of 6–9 mice per group. Control mice were used as a reference and consisted of 3 uninfected mice, pre-treated with a single dose of 5 mg/100 µl vancomycin, and euthanized 3 days post-treatment. WT (B6) mice did not exhibit any significant signs of inflammation, while <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice showed a significant increase relative to the uninfected <i>Sigirr^−/−</i> control, both 3 and 7 DPI. <i>Tlr4^−/−/Sigirr^−/−</i> mice showed a statistically significant increase, relative to control mice at 3 DPI only, but even at 3 DPI, were significantly less than either <i>Sigirr^−/−</i> and <i>Tlr2^−/−/Sigirr^−/−</i> mice. Statistical significance was determined using a two-way ANOVA and a Bonferroni post-test (NS p>0.05, *p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001).</p

Immunofluorescence staining of <i>C. jejuni</i> cecal colonization <i>in vivo</i>.

<p>Formalin-fixed tissue sections of ceca obtained from <i>C. jejuni</i>-infected WT and <i>Sigirr^−/−</i> mice 7 DPI at ×200 magnification. Cell nuclei are stained with DAPI (blue), epithelial cells are outlined with antibodies specific to β-actin (green), and <i>C. jejuni</i> (red) are clearly visible around the edge of the lumen and into the cecal crypts.</p