Defining the effect and mediators of two knowledge translation strategies designed to alter knowledge, intent and clinical utilization of rehabilitation outcome measures: a study protocol [NCT00298727]

BACKGROUND: A substantial number of valid outcome measures have been developed to measure health in adult musculoskeletal and childhood disability. Regrettably, national initiatives have merely resulted in changes in attitude, while utilization remains unacceptably low. This study will compare the effectiveness and mediators of two different knowledge transfer (KT) interventions in terms of their impact on changing knowledge and behavior (utilization and clinical reasoning) related to health outcome measures. METHOD/DESIGN: Physical and occupational therapists (n = 144) will be recruited in partnership with the national professional associations to evaluate two different KT interventions with the same curriculum: 1) Stakeholder-Hosted Interactive Problem-Based Seminar (SHIPS), and 2) Online Problem-Based course (e-PBL). SHIPS will consist of face-to-face problem-based learning (PBL) for 2 1/2 days with outcome measure developers as facilitators, using six problems generated in consultation with participants. The e-PBL will consist of a 6-week web-based course with six generic problems developed by content experts. SHIPS will be conducted in three urban centers in Canada. Participants will be block-allocated by a minimization procedure to either of the two interventions to minimize any prognostic differences. Trained evaluators at each site will conduct chart audits and chart-stimulated recall. Trained interviewers will conduct semi-structured interviews focused on identifying critical elements in KT and implementing practice changes. Interviews will be transcribed verbatim. Baseline predictors including demographics, knowledge, attitudes/barriers regarding outcome measures, and Readiness to Change will be assessed by self-report. Immediately post-intervention and 6 months later, these will be re-administered. Primary qualitative and quantitative evaluations will be conducted 6-months post-intervention to assess the relative effectiveness of KT interventions and to identify elements that contribute to changing clinical behavior. Chart audits will determine the utilization of outcome measures (counts). Incorporation of outcome measures into clinical reasoning will be assessed using an innovative technique: chart-stimulated recall. DISCUSSION: A strategy for optimal transfer of health outcome measures into practice will be developed and shared with multiple disciplines involved in primary and specialty management of musculoskeletal and childhood disability

Listeria pathogenesis and molecular virulence determinants

The gram-positive bacterium Listeria monocytogenes is the causative agent of listeriosis, a highly fatal opportunistic foodborne infection. Pregnant women, neonates, the elderly, and debilitated or immunocompromised patients in general are predominantly affected, although the disease can also develop in normal individuals. Clinical manifestations of invasive listeriosis are usually severe and include abortion, sepsis, and meningoencephalitis. Listeriosis can also manifest as a febrile gastroenteritis syndrome. In addition to humans, L. monocytogenes affects many vertebrate species, including birds. Listeria ivanovii, a second pathogenic species of the genus, is specific for ruminants. Our current view of the pathophysiology of listeriosis derives largely from studies with the mouse infection model. Pathogenic listeriae enter the host primarily through the intestine. The liver is thought to be their first target organ after intestinal translocation. In the liver, listeriae actively multiply until the infection is controlled by a cell-mediated immune response. This initial, subclinical step of listeriosis is thought to be common due to the frequent presence of pathogenic L. monocytogenes in food. In normal indivuals, the continual exposure to listerial antigens probably contributes to the maintenance of anti-Listeria memory T cells. However, in debilitated and immunocompromised patients, the unrestricted proliferation of listeriae in the liver may result in prolonged low-level bacteremia, leading to invasion of the preferred secondary target organs (the brain and the gravid uterus) and to overt clinical disease. L. monocytogenes and L. ivanovii are facultative intracellular parasites able to survive in macrophages and to invade a variety of normally nonphagocytic cells, such as epithelial cells, hepatocytes, and endothelial cells. In all these cell types, pathogenic listeriae go through an intracellular life cycle involving early escape from the phagocytic vacuole, rapid intracytoplasmic multiplication, bacterially induced actin-based motility, and direct spread to neighboring cells, in which they reinitiate the cycle. In this way, listeriae disseminate in host tissues sheltered from the humoral arm of the immune system. Over the last 15 years, a number of virulence factors involved in key steps of this intracellular life cycle have been identified. This review describes in detail the molecular determinants of Listeria virulence and their mechanism of action and summarizes the current knowledge on the pathophysiology of listeriosis and the cell biology and host cell responses to Listeria infection. This article provides an updated perspective of the development of our understanding of Listeria pathogenesis from the first molecular genetic analyses of virulence mechanisms reported in 1985 until the start of the genomic era of Listeria research

Engineering an Enhanced EGFR Engager: Humanization of Cetuximab for Improved Developability

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers such as colorectal cancer and head and neck cancer. Cetuximab is a chimeric monoclonal antibody containing mouse variable regions that bind to EGFR and prevent it from signaling. Although cetuximab has been used clinically since 2004 to successfully control solid tumors, advances in protein engineering have created the opportunity to address some of its shortcomings. In particular, the presence of mouse sequences could contribute to immunogenicity in the form of anti-cetuximab antibodies, and an occupied glycosylation site in FR3 can contribute to hypersensitivity reactions and product heterogeneity. Using simple framework graft or sequence-/structure-guided approaches, cetuximab was humanized onto 11 new frameworks. In addition to increasing humanness and removing the VH glycosylation site, dynamic light scattering revealed increases in stability, and bio-layer interferometry confirmed minimal changes in binding affinity, with patterns emerging across the humanization method. This work demonstrates the potential to improve the biophysical and clinical properties of first-generation protein therapeutics and highlights the advantages of computationally guided engineering

Engineering an Enhanced EGFR Engager: Humanization of Cetuximab for Improved Developability

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose proliferative effects can contribute to the development of many types of solid tumors when overexpressed. For this reason, EGFR inhibitors such as cetuximab can play an important role in treating cancers such as colorectal cancer and head and neck cancer. Cetuximab is a chimeric monoclonal antibody containing mouse variable regions that bind to EGFR and prevent it from signaling. Although cetuximab has been used clinically since 2004 to successfully control solid tumors, advances in protein engineering have created the opportunity to address some of its shortcomings. In particular, the presence of mouse sequences could contribute to immunogenicity in the form of anti-cetuximab antibodies, and an occupied glycosylation site in FR3 can contribute to hypersensitivity reactions and product heterogeneity. Using simple framework graft or sequence-/structure-guided approaches, cetuximab was humanized onto 11 new frameworks. In addition to increasing humanness and removing the VH glycosylation site, dynamic light scattering revealed increases in stability, and bio-layer interferometry confirmed minimal changes in binding affinity, with patterns emerging across the humanization method. This work demonstrates the potential to improve the biophysical and clinical properties of first-generation protein therapeutics and highlights the advantages of computationally guided engineering

Kinetic mechanism of controlled Fab-arm exchange for the formation of bispecific immunoglobulin G1 antibodies

Bispecific antibodies (bsAbs) combine the antigen specificities of two distinct Abs and demonstrate therapeutic promise based on novel mechanisms of action. Among the many platforms for creating bsAbs, controlled Fab-arm exchange (cFAE) has proven useful based on minimal changes to native Ab structure and the simplicity with which bsAbs can be formed from two parental Abs. Despite a published protocol for cFAE and its widespread use in the pharmaceutical industry, the reaction mechanism has not been determined. Knowledge of the mechanism could lead to improved yields of bsAb at faster rates as well as foster adoption of process control. In this work, a combination of Forster resonance energy transfer (FRET), nonreducing SDS-PAGE, and strategic mutation of the Ab hinge region was employed to identify and characterize the individual steps of cFAE. Fluorescence correlation spectroscopy (FCS) was used to determine the affinity of parental (homodimer) and bispecific (heterodimer) interactions within the C(H)3 domain, further clarifying the thermodynamic basis for bsAb formation. The result is a clear sequence of events with rate constants that vary with experimental conditions, where dissociation of the K409R parental Ab into half-Ab controls the rate of the reactio