Recommendations from a Canadian Delphi consensus study on best practice for optimal referral and appropriate management of severe asthma.

BACKGROUND: In Canada, severe asthma affects an estimated 5-10% of people with asthma and is associated with frequent exacerbations, poor symptom control and significant morbidity from the disease itself, as well as the high dose inhaled, and systemic steroids used to treat it. Significant heterogeneity exists in service structure and patient access to severe asthma care, including access to biologic treatments. There appears to be over-reliance on short-acting beta agonists and frequent oral corticosteroid use, two indicators of uncontrolled asthma which can indicate undiagnosed or suboptimally treated severe asthma. The objective of this modified Delphi consensus project was to define standards of care for severe asthma in Canada, in areas where the evidence is lacking through patient and healthcare professional consensus, to complement forthcoming guidelines. METHODS: The steering group of asthma experts identified 43 statements formed from eight key themes. An online 4-point Likert scale questionnaire was sent to healthcare professionals working in asthma across Canada to assess agreement (consensus) with these statements. Consensus was defined as high if ≥ 75% and very high if ≥ 90% of respondents agreed with a statement. RESULTS: A total of 150 responses were received from HCPs including certified respiratory educators, respirologists, allergists, general practitioners/family physicians, nurses, pharmacists, and respiratory therapists. Consensus amongst respondents was very high in 37 (86%) statements, high in 4 (9%) statements and was not achieved in 2 (5%) statements. Based on the consensus scores, ten key recommendations were proposed. These focus on referrals from primary and secondary care, accessing specialist asthma services, homecare provision for severe asthma patients and outcome measures. CONCLUSIONS: Implementation of these recommendations across the severe asthma care pathway in Canada has the potential to improve outcomes for patients through earlier detection of undiagnosed severe asthma, reduction in time to severe asthma diagnosis, and initiation of advanced phenotype specific therapies.

A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9.

ACE2, the first known human homologue of angiotensin-converting enzyme (ACE), was identified from 5' sequencing of a human heart failure ventricle cDNA library. ACE2 has an apparent signal peptide, a single metalloprotease active site, and a transmembrane domain. The metalloprotease catalytic domains of ACE2 and ACE are 42% identical, and comparison of the genomic structures indicates that the two genes arose through duplication. In contrast to the more ubiquitous ACE, ACE2 transcripts are found only in heart, kidney, and testis of 23 human tissues examined. Immunohistochemistry shows ACE2 protein predominantly in the endothelium of coronary and intrarenal vessels and in renal tubular epithelium. Active ACE2 enzyme is secreted from transfected cells by cleavage N-terminal to the transmembrane domain. Recombinant ACE2 hydrolyzes the carboxy terminal leucine from angiotensin I to generate angiotensin 1-9, which is converted to smaller angiotensin peptides by ACE in vitro and by cardiomyocytes in culture. ACE2 can also cleave des-Arg bradykinin and neurotensin but not bradykinin or 15 other vasoactive and hormonal peptides tested. ACE2 is not inhibited by lisinopril or captopril. The organ- and cell-specific expression of ACE2 and its unique cleavage of key vasoactive peptides suggest an essential role for ACE2 in the local renin-angiotensin system of the heart and kidney. The full text of this article is available at http://www. circresaha.org.

Structural and functional characterization of an epitope in the conserved C-terminal region of HIV-1 gp120.

Through an integrated study of the reactivity of a monoclonal antibody, 803-15.6, with synthetic peptides and native recombinant HIV-1 envelope glycoprotein gp120, we have obtained structure-functional information on a region of rgp120 not yet elucidated by X-ray crystallography. mAb 803-15.6 binds with high affinity and broad cross-clade specificity to the conserved C-terminal region (amino acids 502-516) of HIV-1 rgp120. Phage display selection from a random peptide library identified the core binding motif as AXXKXRH, homologous to residues 502-508. Using quantitative binding analyses, the affinity of mAb 803-15.6 for native, monomeric recombinant gp120HXB2 (rgp120) was found to be similar to that for the synthetic gp120 peptide (502-516). Circular dichroism studies indicate that the synthetic peptide largely has a random coil conformation in solution. The results therefore suggest that the 803-15.6 epitope is fully accessible on rgp120 and that this region of rgp120 is as flexible as the synthetic peptide. Residues 502-504 are on the edge of a putative gp41 binding site that has been postulated to change conformation on CD4 binding. However, the affinity of mAb 803-15.6 for rgp120 is not affected by binding of CD4 and vice-versa. These results suggest either that the 502-504 region does not change conformation upon CD4 binding, or that recombinant gp120 does not undergo the same changes as occur in the native viral gp120-gp41 oligomer. The detailed characterization of the 803-15.6 epitope may be useful for further study of the role of the C5 region of gp120 in the viral attachment and fusion process.

Construction and characterization of a radio-iodinatable mutant of recombinant human CD4.

Recombinant soluble human CD4 (rsCD4) has been used in iodinated form to study the interaction of CD4 with its ligands. However, the utility of [125I]-rsCD4 is limited because rsCD4 is inefficiently iodinated and the iodinated protein is poorly active. The iodination properties of rsCD4 most likely reflect the poor accessibility of the tyrosine residues, apparent from the available X-ray structures. We have generated an iodinatable mutant of rsCD4 by substituting Tyr for Phe(179) in the flexible, solvent-exposed C-terminal region of rsCD4(183), a truncated form of CD4 that consists of the first 183 residues of CD4 and includes the binding sites for HIV-1 gp120 and MHC class II molecules. When F179Y rsCD4(183) is iodinated under trace-labeling conditions, the efficiency of 125I incorporation and the percentage of iodinated molecules that are active are much enhanced compared with WT rsCD4. Moreover, trace-labeled [125I]-F179Y rsCD4(183) has the same affinity for HIV-1 rgp120 as unlabeled WT rsCD4. The improved activity of trace-labeled [125I]-F179Y rsCD4(183) appears to be due to effective competition by Y179 for reactive iodine species that, in WT rsCD4, react with traces of denatured protein and/or with residues critical for activity or conformational integrity. The incorporation of accessible tyrosine residues may improve the iodinatibility of a protein both by introducing a readily iodinatable residue and by protecting sensitive proteins from adverse reactions.