Upregulation of CD8+ regulatory T cells following liver-directed AAV gene therapy.

Liver-directed AAV gene therapy represents a unique treatment modality for a host of diseases. This is due, in part, to the induction of tolerance to transgene products. Despite the plethora of recognized regulatory cells in the body, there is currently a lack of literature supporting the induction of non-CD4+ regulatory cells following hepatic AAV gene transfer. In this work, we show that CD8+ regulatory T cells are up-regulated in PBMCs of mice following capsid only and therapeutic transgene AAV administration. Further, we demonstrate that hepatic AAV gene transfer results in a significant increase in CD8+ regulatory T cells following experimental autoimmune encephalomyelitis induction. Notably, this response occurred only in therapeutic vector treated animals, not capsid only controls. Understanding the role these cells play in treatment efficacy will result in the development of improved AAV vectors that take advantage of the full gamut of regulatory cells within the body.

Induction of antigen-specific tolerance by hepatic AAV immunotherapy regardless of T cell epitope usage or mouse strain background.

In vivo induction of antigen (Ag)-specific regulatory T cells (Treg) is considered the holy grail of therapeutic strategies for restoring tolerance in autoimmunity. Unfortunately, in the autoimmune disease multiple sclerosis, an effective and durable therapy targeting the diverse repertoire of emerging Ags without compromising the patient's natural immunity has remained elusive. To address this deficiency, we have developed an Ag-specific adeno-associated virus (AAV) immunotherapy that will restore tolerance in a Treg-dependent manner. Using multiple strains of mice with different genetic and immunological backgrounds, we demonstrate that a liver directed AAV vector expressing a single transgene can prevent experimental autoimmune encephalomyelitis from developing and effectively mitigate pre-existing or established disease that was induced by one or more auto-reactive myelin oligodendrocyte glycoprotein-derived peptides. Overall, the results suggests that AAV can efficiently restore Ag-specific immune tolerance to an immunogenic protein that is neither restricted by the major histocompatibility complex haplotype, nor by the specific antigenic epitope(s) presented. These findings may pave the way for developing a comprehensive Ag-specific immunotherapy that does not require prior knowledge of the specific immunogenic epitopes and that may prove to be universally applicable to all MS patients, and adaptable for other autoimmune diseases.

Oral Pathogen Porphyromonas gingivalis Can Escape Phagocytosis of Mammalian Macrophages.

Macrophages are phagocytic cells that play a key role in host immune response and clearance of microbial pathogens. Porphyromonas gingivalis is an oral pathogen associated with the development of periodontitis. Escape from macrophage phagocytosis was tested by infecting THP-1-derived human macrophages and RAW 264.7 mouse macrophages with strains of P. gingivalis W83 and 33277 as well as Streptococcus gordonii DL1 and Escherichia coli OP50 at MOI = 100. CFU counts for all intracellular bacteria were determined. Then, infected macrophages were cultured in media without antibiotics to allow for escape and escaping bacteria were quantified by CFU counting. P. gingivalis W83 displayed over 60% of the bacterial escape from the total amount of intracellular CFUs, significantly higher compared to all other bacteria strains. In addition, bacterial escape and re-entry were also tested and P. gingivalis W83, once again, showed the highest numbers of CFUs able to exit and re-enter macrophages. Lastly, the function of the PG0717 gene of P. gingivalis W83 was tested on escape but found not related to this activity. Altogether, our results suggest that P. gingivalis W83 is able to significantly avoid macrophage phagocytosis. We propose this ability is likely linked to the chronic nature of periodontitis.

Age-adjusted D-dimer thresholds in the investigation of suspected pulmonary embolism: A retrospective evaluation in patients ages 50 and older using administrative data.

OBJECTIVES: D-dimer testing is an important component of the workup for pulmonary embolism (PE). However, age-related increases in D-dimer concentrations result in false positives in older adults, leading to potentially unnecessary imaging utilization. The objective of this study was to quantify the test characteristics of an age-adjusted D-dimer cut-off for ruling out PE in older patients investigated in actual clinical practice. METHODS: This observational study used administrative data from four emergency departments from July 2013 to January 2015. Eligible patients were ages 50 and older with symptoms of PE who underwent D-dimer testing. The primary outcome was 30-day diagnosis of PE, confirmed by imaging reports. Test characteristics of the D-dimer assay were calculated using the standard reference value (500 ng/ml), the local reference value (470 ng/ml), and an age-adjusted threshold (10 ng/ml × patient's age). RESULTS: This cohort includes 6,655 patients ages 50 and older undergoing D-dimer testing for a possible PE. Of these, 246 (3.7%) were diagnosed with PE. Age-adjusted D-dimer cut-offs were more specific than standard cut-offs (75.4% v. 63.8%) but less sensitive (90.3% v. 97.2%). The false-negative risk in this population was 0.49% using age-adjusted D-dimer cut-offs compared with 0.15% with traditional cut-offs. CONCLUSION: Age-adjusted D-dimer cut-offs are substantially more specific than traditional cut-offs and may reduce CT utilization among older patients with suspected PE. We observed a loss of sensitivity, with an increased risk of false-negatives, using age-adjusted cut-offs. We encourage further evaluation of the safety and accuracy of age-adjusted D-dimer cut-offs in actual clinical practice.

Limitations of pulmonary embolism ICD-10 codes in emergency department administrative data: let the buyer beware.

BACKGROUND: Administrative data is a useful tool for research and quality improvement; however, validity of research findings based on these data depends on their reliability. Diagnoses assigned by physicians are subsequently converted by nosologists to ICD-10 codes (International Statistical Classification of Diseases and Related Health Problems, 10th Revision). Several groups have reported ICD-9 coding errors in inpatient data that have implications for research, quality improvement, and policymaking, but few have assessed ICD-10 code validity in ambulatory care databases. Our objective was to evaluate pulmonary embolism (PE) ICD-10 code accuracy in our large, integrated hospital system, and the validity of using these codes for operational and health services research using ED ambulatory care databases. METHODS: Ambulatory care data for patients (age ≥ 18 years) with a PE ICD-10 code (I26.0 and I26.9) were obtained from the records of four urban EDs between July 2013 to January 2015. PE diagnoses were confirmed by reviewing medical records and imaging reports. In cases where chart diagnosis and ICD-10 code were discrepant, chart review was considered correct. Physicians' written discharge diagnoses were also searched using 'pulmonary embolism' and 'PE', and patients who were diagnosed with PE but not coded as PE were identified. Coding discrepancies were quantified and described. RESULTS: One thousand, four hundred and fifty-three ED patients had a PE ICD-10 code. Of these, 257 (17.7%) were false positive, with an incorrectly assigned PE code. Among the 257 false positives, 193 cases had ambiguous ED diagnoses such as 'rule out PE' or 'query PE', while 64 cases should have had non-PE codes. An additional 117 patients (8.90%) with a PE discharge diagnosis were incorrectly assigned a non-PE ICD-10 code (false negative group). The sensitivity of PE ICD-10 codes in this dataset was 91.1% (95%CI, 89.4-92.6) with a specificity of 99.9% (95%CI, 99.9-99.9). The positive and negative predictive values were 82.3% (95%CI, 80.3-84.2) and 99.9% (95%CI, 99.9-99.9), respectively. CONCLUSIONS: Ambulatory care data, like inpatient data, are subject to coding errors. This confirms the importance of ICD-10 code validation prior to use. The largest proportion of coding errors arises from ambiguous physician documentation; therefore, physicians and data custodians must ensure that quality improvement processes are in place to promote ICD-10 coding accuracy.

The theory of planned behaviour explains intentions to use antiresorptive medication after a fragility fracture.

Our objective was to ascertain whether the Theory of Planned Behaviour (TPB) explains patient intentions to use antiresorptive medication after a fracture. A qualitative study was conducted with English-speaking members of the Canadian Osteoporosis Patient Network (COPN) who had sustained a fragility fracture at 50+ years of age and were not taking antiresorptive medication at the time of that fracture. Questions during a 1-h telephone interview were guided by the domains of the TPB: they addressed the antecedent constructs regarding antiresorptive medication (attitudes, subjective norms, and perceived behavioural control) as well as intentions regarding antiresorptive medication use. We created a coding template a priori based on the TPB domains and applied this template to the interview data. Twenty-six eligible participants (24 females, 2 males) aged 51-89 completed an interview. The TPB appeared to be predictive of intentions in 19 (73%) participants. In the majority of participants where the TPB did not appear to be predictive (57%), a positive attitude toward antiresorptive medication was the most important antecedent variable in determining intentions. The TPB appeared to be predictive of intentions to use antiresorptive medication among individuals who had experienced a fragility fracture. Attitudes towards medication were especially important.

What Do We Know about Individuals Who Are Assessed as Being at Moderate Risk for Future Fracture in Canada?

OBJECTIVE: We examined what was known about individuals in Canada who were assessed as being at moderate risk for future fracture. METHODS: A scoping review was conducted. Eligible articles were Canadian studies published from 2010 onwards reporting on primary data that included patients at moderate risk for future fracture. We limited the search to Canada as fracture risk categorization is unique to each country. Studies were identified by searching relevant databases. Two reviewers independently reviewed titles and abstracts to determine each study's eligibility. General information about each study, demographic information about the moderate risk groups (including tool used to determine moderate risk (Fracture Risk Assessment Tool (FRAX), Canadian Association of Radiologists and Osteoporosis Canada (CAROC)), and outcomes (number of patients: recommended treatment, prescribed treatment, initiating treatment, persisting with treatment after six months, who re-fractured, who died) were documented. RESULTS: We identified 1193 papers which were further screened for eligibility. Of the 1193 identified, 7 were eligible for the review but only 4 articles contained demographic or outcome data on moderate risk patients. In one study, 1.8% of moderate risk patients died over a mean 5.3 years of observation and in three studies, the risk of fracture was 5.9% over a median of 3 years of follow-up, 8.3% over a mean of 5.4 years, and 14.7% over 10 years of follow-up. CONCLUSION: There is a wide knowledge gap in the literature concerning individuals who are assessed as moderate risk for future fracture in Canada.