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.

Role of omalizumab in insulin hypersensitivity: a case report and review of the literature.

BACKGROUND: Insulin allergy is a rare yet severe side effect of exogenous insulin use. Management typically involves use of alternative antihyperglycaemic agents, symptom control with antihistamines, use of different insulin formulations, and induction of tolerance with incremental doses of insulin. This treatment regimen is not always successful, and the use of omalizumab, an anti-IgE monoclonal antibody, has been used to induce tolerance to insulin. CASE REPORT: G.M. is a 62-year-old man with Type 2 diabetes mellitus. His condition was not optimized on oral agents, and insulin therapy was required. G.M. had anaphylaxis to insulin NPH, and subsequent skin-prick testing was positive to insulin aspart, insulin NPH, insulin glulisine, insulin detemir, regular insulin, insulin glargine 100 units/ml and insulin glargine 300 units/ml. He received incremental doses of several insulin formulations; however, he experienced diffuse urticaria preventing optimal glycaemic control. Three successful cases have been described in the literature of omalizumab inducing tolerance to exogenous insulin; therefore, G.M. was started on omalizumab. He subsequently tolerated treatment doses of insulin glulisine and insulin detemir with no allergic reactions and with improvement in glycaemic control. CONCLUSION: To our knowledge, this is the first described case of allergy to insulin glargine 300 units/ml and reiterates the potential use of omalizumab in insulin allergy. Further research is warranted to determine if omalizumab should be considered standard of care in difficult-to-treat insulin hypersensitivity.

Long-term efficacy and safety of infliximab maintenance therapy in patients with plaque-type psoriasis in real-world practice.

BACKGROUND: Tumour necrosis factor-α inhibitors, including infliximab (IFX), can improve disease control of plaque-type psoriasis. OBJECTIVES: The Real-World Assessment of Long-Term Infliximab Therapy for Psoriasis (REALITY) study evaluated the efficacy and safety of maintenance IFX therapy in typical clinical settings. METHODS: In this prospective, observational, open-label, multicentre study in patients with plaque-type psoriasis, IFX 5 mg kg was infused at weeks 0, 2 and 6, and every 8 weeks thereafter during a 50-week treatment phase. The primary outcome was ≥ 75% Psoriasis Area and Severity Index (PASI) improvement from baseline to week 50. Patients with ≥ 25% PASI improvement from baseline to the end of the treatment phase were potentially eligible to enter a 48-week extended treatment phase. Response maintenance and other efficacy measures were evaluated. Adverse events (AEs) were collected. RESULTS: In total 660 patients enrolled. Of 521 efficacy-evaluable treatment phase patients (66% male, mean age 46·5 years, mean PASI 18·1), 56·8% achieved PASI 75 at the end of the treatment phase. Response was maintained at week 50 by 64·7% (205/317) of patients who achieved PASI 75 at week 14. During extended treatment, 66·3% (112/169) of patients attained PASI 75 at week 98; response was maintained at week 98 by 71·6% (101/141) of those who achieved PASI 75 at week 50. IFX was generally well tolerated. During treatment, 7·6% (50/659) of patients had serious AEs. During extended treatment, 4·1% (eight of 193) of patients had serious AEs. CONCLUSIONS: PASI 75 response was achieved by 56·8% and 66·3% of patients at weeks 50 and 98, respectively. The AE pattern was consistent with previous reports.

Preclinical development of siRNA therapeutics for AL amyloidosis.

Amyloid light chain (AL) amyloidosis is a rare hematologic disorder characterized by the accumulation of a misfolded monoclonal immunoglobulin (Ig) light chain (LC) as fibrillar protein deposits. Current treatments, including cytotoxic chemotherapy and immunomodulatory therapy, are directed at killing the plasma cells that produce the LCs, but have significant toxicity for other cell types. We have designed small interfering RNAs (siRNAs) targeting the amyloidogenic LC messenger RNA (mRNA) in order to reduce expression of the amyloid precursor protein. Using nanomolar concentrations of siRNAs, we have inhibited synthesis of LC in transfected cells in vitro in a dose-dependent fashion. Furthermore, in an in vivo plasmacytoma mouse model of AL amyloidosis, we have demonstrated that these siRNAs can significantly reduce local production and circulating levels of LC. This model system highlights the therapeutic potential of siRNA for AL amyloidosis.

Prescription rates of protective co-therapy for NSAID users at high GI risk and results of attempts to improve adherence to guidelines.

BACKGROUND: Protective co-therapy is recommended in NSAID users with GI risk factors, but adherence is poor. AIM: To assess the proportion of NSAID users receiving co-therapy and strategies to improve adherence. METHODS: Arthritis patients > or =50 years of age received etoricoxib or diclofenac in a double-blind randomized trial. Reminders that high-risk patients (age > or = 65; previous ulcer/haemorrhage; corticosteroid, anticoagulant, aspirin use) should receive co-therapy were given at study initiation. Free PPI was provided. An intervention midway through the study included a written reminder and required written response regarding co-therapy. RESULTS: 16,244/23,504 (69%) patients had GI risk factors. Pre-intervention, co-therapy was most common with previous ulcer/haemorrhage [706/1107 (64%)] and 3-4 risk factors [331/519 (64%)]. In the 10,026 patients enrolled pre-intervention and remaining in the study > or =6 months after, co-therapy in high-risk patients increased from 2958/6843 (43%) to 4177/6843 (61%) (difference = 18%; 95% CI 16%,19%). The increase was greater outside the US (22%; 19%,24%) than in the US (15%; 13%,17%). CONCLUSIONS: Less than 50% of NSAID users with GI risk factors are given protective co-therapy--even if prescribers are given reminders and cost is not an issue. Direct communication requiring written response significantly increased adherence to guidelines, but achieving higher levels of adherence will require additional strategies.

Templated growth of graphenic materials.

A novel strategy is proposed for the topologically controlled synthesis of extended graphenic sheets by additively reacting carbon into a pre-existing graphene sheet which is on top of a templating substrate. This concept is implemented and demonstrated using chemical vapor deposition (CVD). Novel morphological features observed in this study suggest unusual aspects of the CVD growth process. CVD results demonstrate the basic soundness of the synthesis strategy but highlight the sensitivity of the process to certain types of disruption and the need for alternative forms of embodiment.

Fine-scale population differentiation and gene flow in a terrestrial salamander (Plethodon cinereus) living in continuous habitat.

Several recent studies have shown that amphibian populations may exhibit high genetic subdivision in areas with recent fragmentation and urban development. Less is known about the potential for genetic differentiation in continuous habitats. We studied genetic differentiation of red-backed salamanders (Plethodon cinereus) across a 2-km transect through continuous forest in Virginia, USA. Mark-recapture studies suggest very little dispersal for this species, whereas homing experiments and post-Pleistocene range expansion both suggest greater dispersal abilities. We used six microsatellite loci to examine genetic population structure and differentiation between eight subpopulations of red-backed salamanders at distances from 200 m to 2 km. We also used several methods to extrapolate dispersal frequencies and test for sex-biased dispersal. We found small, but detectable differentiation among populations, even at distances as small as 200 m. Differentiation was closely correlated with distance and both Mantel tests and assignment tests were consistent with an isolation-by-distance model for the population. Extrapolations of intergenerational variance in spatial position (sigma(2)<15 m(2)) and pair-wise dispersal frequencies (4 Nm < 25 for plots separated by 300 m) both suggest limited gene flow. Additionally, tests for sex-biased dispersal imply that dispersal frequency is similarly low for both sexes. We suggest that these low levels of gene flow and the infrequent dispersal observed in mark-recapture studies may be reconciled with homing ability and range expansion if dispersing animals rarely succeed in breeding in saturated habitats, if dispersal is flexible depending on the availability of habitat, or if dispersal frequency varies across the geographic range of red-backed salamanders.

Infusion of light chains from patients with cardiac amyloidosis causes diastolic dysfunction in isolated mouse hearts.

BACKGROUND: Primary (AL) amyloidosis is a plasma cell dyscrasia characterized by clonal production of immunoglobulin light chains (LC) resulting in the subsequent systemic deposition of extracellular amyloid fibrils. Cardiac involvement is marked by the hemodynamic pattern of impaired diastolic filling and restrictive cardiomyopathy. Although cardiac death in patients with AL amyloidosis is usually associated with extensive myocardial infiltration, the infiltration alone does not correlated with the degree of heart failure or survival. We hypothesized that circulating monoclonal LC may directly impair cardiac function, in addition to any mechanical effects of amyloid fibril deposition. Therefore, we examined the effects of amyloid LC proteins on diastolic and systolic cardiac function, as measured in an isolated mouse heart model. METHODS AND RESULTS: LC were obtained from patients with nonamyloid disease or from those with noncardiac, mild cardiac, and severe cardiac involved AL amyloidosis. Saline or LC (100 microgram/mL) was infused into a Langendorff-perfused, isovolumically contracting mouse heart. Saline and control, noncardiac, and mild-cardiac LC infusions did not alter ex vivo cardiac function. In contrast, infusion of sever cardiac LC resulted in marked impairment of ventricular relaxation with preservation of contractile function. CONCLUSION: These results demonstrate that infusion of LC from patients with AL amyloidosis result in diastolic dysfunction similar to that observed in patients with cardiac involved AL amyloidosis, and they suggest that amyloid LC proteins may contribute directly to the pathogenesis and the rapid progression of amyloid cardiomyopathy, independent of extracellular fibril deposition.

Biophysical analysis of normal transthyretin: implications for fibril formation in senile systemic amyloidosis.

Transthyretin (TTR) is a plasma protein that transports thyroid hormone and retinol binding protein-vitamin A complex. Eighty-four variants of TTR have been identified and seventy-four are associated with familial amyloidotic polyneuropathy. Normal TTR is the major protein found in the fibrillar deposits in the heart at time of autopsy of individuals with senile systemic amyloidosis. The mechanism by which normally soluble TTR deposits as organ-damaging, insoluble, pathological fibrils late in life is unknown. Understanding the mechanism of fibrillogenesis of normal TTR is critical to the design of clinical treatments aimed at retardation, prevention, or reversal of fibril deposition. We have employed a biophysical approach to explore the hypothesis that an instability in a particular secondary or tertiary structure plays a role in the ability of normal TTR to form fibrils at physiological pH. Using far UV circular dichroic (CD) spectroscopy as a function of temperature we have identified simultaneous, cooperative, reversible structural changes in the beta-sheet and alpha-helical regions. The flexible short, surface-located loops undergo an irreversible conformational change at a lower temperature. Spectra before and after heating are different, particularly in the wavelength region associated with these loops, strongly suggesting that the major portion of TTR returns to its initial conformation while the loops do not. Near UV CD reveals partially reversible and irreversible changes in tertiary structure. Using calorimetry to directly measure the enthalpy associated with these changes, two peaks are observed, with further analysis suggesting conformational intermediates. Precipitates from heated samples reveal pre-fibrillar morphology by negative stain electron microscopy. These biophysical studies suggest that heat-induced conformational rearrangements enable normal TTR to assemble into pre-fibrils at physiological pH.

Changing patterns of presentations of patients with HIV-related disease at a tertiary referral centre and its implications for physician training.

Highly active antiretroviral therapy (HAART) has been shown to be highly effective in controlling HIV-related disease progression. Our objective was to determine whether HAART had altered the spectrum of HIV-related disease presentations at a tertiary medical referral centre and if a change in the clinical presentations of HIV-infected individuals to the hospital had impacted on physicians' training. A retrospective study which examined all admissions of HIV-infected patients identified between 1 October 1996 to 30 September 1998 using a hospital-designed computer database was undertaken at the Beth Israel Deaconess Medical Center (BIDMC) tertiary medical referral centre. All medical residents were surveyed in order to assess their knowledge of HIV-associated admissions and their confidence treating HIV-infected patients. There were significant changes in the admitting diagnosis for HIV-related illness between 1996 and 1998. Admissions for opportunistic infections (OIs) declined whereas admissions with bacterial infections increased significantly. Use of HAART remained stable between the 2 years of the study. Physicians' overestimated the use of HAART and only 8% of residents felt very comfortable taking care of an HIV-infected patient. In conclusion, the spectrum of presentations with HIV-related disease to a tertiary referral centre continues to change in the HAART era and impacts on physicians' experience of the management of HIV disease.

Betabellins 15D and 16D, de Novo designed beta-sandwich proteins that have amyloidogenic properties.

The betabellin structure is a de novo designed beta-sandwich protein consisting of two 32-residue beta-sheets packed against one another by hydrophobic interactions. d-Amino acid residues are used to energetically favor formation of type-I' beta turns. Air oxidation of betabellin 15S (B15S) (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p denotes d-Pro, h denotes d-His, and k denotes d-Lys) yields betabellin 15D (B15D), a 64-residue disulfide-bridged protein. The amino acid sequence of B15D contains a conformationally constrained d-Pro residue at the i + 1 position of each type-I' beta turn. To test whether d-Pro residues are necessary for folding at these positions, the six d-Pro residues of B15D are replaced by d-Ala residues in betabellin 16D (B16D). Previously, transmission electron microscopy showed that B15D forms unbranched, 35-A wide fibrils that associate into bundles in 5.0 mM 3-(N-morpholino)propanesulfonate and 250 mM NaCl at pH 7; under these conditions, B16D forms ribbon-like assemblies. The B15D fibrils resemble the protofilaments that constitute amyloid fibrils. The present studies show that both B15D and B16D have characteristics of amyloidogenic proteins: the unbranched fibrils and ribbons stained with Congo red and displayed a green birefringence, exhibited a cross-beta structure, and bound 1-anilino-8-naphthalenesulfonate. Thus, these de novo designed beta-sandwich proteins should provide useful models for studying the mechanism of amyloid protofilament formation and assembly into amyloid fibrils and for designing potential inhibitors of amyloidogenesis.

Detection of transthyretin variants using immunoprecipitation and matrix-assisted laser desorption/ionization bioreactive probes: a clinical application of mass spectrometry.

In our continuing efforts to develop mass spectrometry-based methods for transthyretin (TTR) variant detection and characterization, we have sought to use matrix-assisted laser desorption/ionization (MALDI) bioreactive probes incorporating immobilized trypsin for screening purposes. These devices show good diagnostic potential as a clinical screening tool to detect amino acid substitutions in TTR. MALDI probes allow the on-probe generation of tryptic digests. The subsequent mass analysis of the on-probe digest yields the peptide map. The inherent advantages of this method include considerably reduced digestion times (minutes vs. hours), absence of autolysis products, minimized sample handling, and hence minimal sample loss. A further advantage is that the opportunity for loss of hydrophobic peptides is reduced because no sample transfer occurs. The method can be applied as a preliminary screen for TTR variants where TTR is isolated from patient serum through immunoprecipitation. This method should also be applicable to other proteins and suitable for automation.

A new transthyretin variant (Ser23Asn) associated with familial amyloidosis in a Portuguese patient.

The detection and characterization of a new transthyretin (ATTR) variant, Ser23Asn, associated with cardiomyopathy in a Portuguese patient with familial amyloidosis is described. Isoelectric focusing (IEF) of serum from the propositus demonstrated heterozygosity for the presence of wild type and variant ATTR. A combination of mass spectrometric (MS) analyses, including electrospray ionization mass spectrometry (ESI MS), high performance liquid chromatography (HPLC)/ESI MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) performed on the serum-derived TTR were used to identify and locate the amino acid replacement in the variant protein. Genetic mutation analysis by DNA sequencing and allele-specific PCR confirmed this finding.

Identification of a novel substitution in the constant region of a gene coding for an amyloidogenic kappa1 light chain.

Current concepts regarding the association between immunoglobulin (Ig) light chain structure and AL amyloidosis (AL) emphasize Ig variable region amino acid substitutions because the majority of light chain amyloid fibrils that have been sequenced contain amino termini of the variable region with only small amounts of the constant region. In this report, we describe a patient with rapidly progressive AL whose amyloid deposits contained primarily monoclonal kappa light chain constant region fragments. We sequenced and analyzed this AL protein, determining that it was an O18-O8 kappa1 variant and that the constant region possessed an unusual Ser-->Asn substitution at position 177. Using pre-mortem bone marrow cells, we cloned and sequenced the cDNA for this AL protein (HCAK1) and, using DNA from post-mortem somatic tissue, we cloned and sequenced the patient's kappa germline O18-O8 donor and kappa constant region (Ckappa) gene segments. The cDNA that coded for HCAK1 contained a variable region that was derived from O18-O8, showing 96.1% homology to germline, and a Ckappa that had a nucleotide substitution (AGC to AAC), resulting in the 177Ser-->Asn replacement. Two Ckappa genes were cloned from somatic tissue DNA, one identical to a known Ckappa sequence and another containing this substitution which likely is a new Ckappa allotype. Our findings indicate that further investigation is warranted into the contributions genetic polymorphisms and light chain constant regions may make to amyloidogenesis.

The use of plasma HIV RNA as a study endpoint in efficacy trials of antiretroviral drugs.

OBJECTIVES: To evaluate the utility of HIV RNA as an endpoint in antiretroviral efficacy studies. DESIGN: Data collected from antiretroviral efficacy trials were analyzed to explore relationships between clinical progression and the magnitude, nadir and duration of HIV RNA reductions. The proportion of patients suppressing HIV RNA below assay quantification, time to maximal virologic response, and loss of virologic response in relation to pretreatment characteristics were also analyzed. METHODS: Analyses were conducted using data from individual antiretoviral efficacy trials or groups of trials that studied similar types of drug regimens and used similar HIV RNA assays. Treatment regimens were pooled for most analyses. Clinical progression was defined as the occurrence of an AIDS-defining event (essentially Centers of Disease Control criteria) or death. RESULTS: Treatment-induced reductions in HIV RNA approximating total assay variability of about 0.5 log10 copies/ml were associated with decreases in the risk of clinical progression. Larger and more sustained reductions in HIV RNA were directly associated with lower risks for disease progression. Lower initial HIV RNA reductions were associated with more durable HIV RNA suppression. CONCLUSIONS: For antiretoviral efficacy studies, plasma HIV RNA is a suitable study endpoint that is likely to predict a decreased risk for AIDS progression and death. Because greater and more sustained reductions in HIV RNA appear to confer greater reductions in clinical risk, maintaining maximal suppression of plasma HIV RNA, particularly below the limits of assay quantification, appears to be a rigorous benchmark for assessing the efficacy of antiretroviral regimens.

A new amyloidogenic transthyretin variant (Val122Ala) found in a compound heterozygous patient.

A new TTR variant, Val122Ala, was characterized in an individual who carried the Gly6Ser polymorphism on the opposite allele. The main clinical feature of this familial transthyretin amyloidosis (ATTR) variant is extensive cardiomyopathy. The detection and characterization of the variant were performed using a combination of isoelectric focusing (IEF), restriction fragment length polymorphism (RFLP), immunoprecipitation, electrospray ionization mass spectrometry (ESIMS), HPLC (high performance liquid chromatography)/ESIMS, and matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS). The results were confirmed by DNA analysis. The propositus has a brother who carries the new variant but not the polymorphism.