Determining the Repertoire of Immunodominant Proteins via Whole-Genome Amplification of Intracellular Pathogens

Culturing many obligate intracellular bacteria is difficult or impossible. However, these organisms have numerous adaptations allowing for infection persistence and immune system evasion, making them some of the most interesting to study. Recent advancements in genome sequencing, pyrosequencing and Phi29 amplification, have allowed for examination of whole-genome sequences of intracellular bacteria without culture. We have applied both techniques to the model obligate intracellular pathogen Anaplasma marginale and the human pathogen Anaplasma phagocytophilum, in order to examine the ability of phi29 amplification to determine the sequence of genes allowing for immune system evasion and long-term persistence in the host. When compared to traditional pyrosequencing, phi29-mediated genome amplification had similar genome coverage, with no additional gaps in coverage. Additionally, all msp2 functional pseudogenes from two strains of A. marginale were detected and extracted from the phi29-amplified genomes, highlighting its utility in determining the full complement of genes involved in immune evasion

Exploring Mechanisms of Change in a Dyadic Relationship Intervention for Siblings in Foster Care

There is increasing recognition of the potential for sibling relationships to promote the well‐being of youth in out‐of‐home care. Efficacious interventions now exist to strengthen the sibling relationships for youth in out‐of‐home care, yet the processes through which these interventions work to enhance sibling relationships remains largely speculative. The current study therefore aimed to identify the critical components of an efficacious dyadic relationship enhancement intervention for siblings in foster care through a secondary analysis of fidelity of implementation and trial outcome data. Data for 168 youth from the treatment condition of the Supporting Siblings in Foster Care study were analysed. Fidelity of implementation was assessed across seven intervention domains: Sibs 4 Life, Positive/Sib Thinking, Activity Planning, Problem Solving, Cooperation, Managing Feelings and Adult Allies. Trial outcome data were drawn from efficacy test results and included a multi‐agent construct of sibling relationship quality. Descriptive statistics detailed intervention implementation, and hierarchical linear models examined associations between intervention coverage, comprehension, and engagement, and 18‐month improvements to the sibling relationship. Results indicate high fidelity to implementation, and Positive/Sib Thinking was the primary intervention domain associated with sibling relationship improvements. Considerations for future mechanisms‐based intervention research with siblings in out‐of‐home care are provided

Neutrophil bactericidal activity against Staphylococcus aureus adherent on biological surfaces. Surface-bound extracellular matrix proteins activate intracellular killing by oxygen-dependent and -independent mechanisms.

The activation patterns of surface adherent neutrophils are modulated via interaction of extracellular matrix proteins with neutrophil integrins. To evaluate neutrophil bactericidal activity, Staphylococcus aureus adherent to biological surfaces were incubated with neutrophils and serum, and the survival of surface bacteria was determined. When compared to albumin-coated surfaces, the bactericidal activity of neutrophils adherent to purified human extracellular matrix was markedly enhanced (mean survival: 34.2% +/- 9.0% of albumin, P less than 0.0001) despite similar efficient ingestion of extracellular bacteria. Enhancement of killing was observed when surfaces were coated with purified constituents of extracellular matrix, i.e., fibronectin, fibrinogen, laminin, vitronectin, or type IV collagen. In addition to matrix proteins, the tetrapeptide RGDS (the sequence recognized by integrins) crosslinked to surface bound albumin was also active (survival: 74.5% +/- 5.5% of albumin, P less than 0.02), and fibronectin-increased killing was inhibited by soluble RGDS. Chemiluminescence measurements and experiments with CGD neutrophils revealed that both oxygen-dependent and -independent bactericidal mechanisms are involved. In conclusion, matrix proteins enhance intracellular bactericidal activity of adherent neutrophils, presumably by integrin recognition of RGDS-containing ligands. These results indicate a role for extracellular matrix proteins in the enhancement of the host defense against pyogenic infections