Local anaesthesia for pain control in first trimester surgical abortion.

BACKGROUND: Abortions prior to 14 weeks are among the most common outpatient surgical procedures performed on people capable of becoming pregnant. Various methods have been used to control pain; however, many people still experience pain with the procedure. OBJECTIVES: To evaluate the benefits and harms of local anaesthesia given for pain control during surgical abortion at less than 14 weeks' gestation. SEARCH METHODS: We searched CENTRAL (Ovid EBM Reviews), MEDLINE (Ovid), Embase, POPLINE, and Google Scholar to December 2022 for randomized controlled trials of pain control in surgical abortion at less than 14 weeks' gestation using suction aspiration. We searched the reference lists of related reviews and articles. SELECTION CRITERIA: We selected effectiveness and comparative effectiveness randomized controlled trials that studied local anaesthesia with common local anaesthetics and administration routes given for pain control in surgical abortion at less than 14 weeks' gestation using uterine aspiration. Outcomes included intraoperative pain, patient satisfaction, and adverse events. DATA COLLECTION AND ANALYSIS: We used standard Cochrane methods. We computed mean differences (MD) with 95% confidence intervals (CI) for continuous variables reporting a mean. We assessed the certainty of evidence using GRADE. MAIN RESULTS: Thirteen studies with 1992 participants met the inclusion criteria. Due to heterogeneity of interventions, we could not pool more than two studies for any outcome. We used 13 mm improvement on a visual/verbal analogue scale to indicate a clinically meaningful difference in pain with surgical abortion (pain with dilation, aspiration, or during procedure). Based on type of pain control, we divided studies into three groups. Paracervical block (PCB) effectiveness trials A 20 mL 1% lidocaine PCB reduced pain with dilation (MD -37.00, 95% CI -45.64 to -28.36), and aspiration (MD -26.00, 95% CI -33.48 to -18.52) compared to a sham PCB (1 RCT, 120 participants; high-certainty evidence). A PCB with 14 mL of 1% chloroprocaine resulted in a slight reduction in pain with aspiration compared to a PCB with normal saline injected at two or four sites (MD -1.50, 95% CI -2.45 to -0.55; 1 RCT, 79 participants; high-certainty evidence). PCB comparative effectiveness trials An ultracaine PCB probably results in little to no clinically meaningful difference in pain during procedure compared to topical cervical lidocaine spray (median 1 point higher, interquartile range (IQR) 0 to 3; P < 0.001; 1 RCT, 48 participants; moderate-certainty evidence). A 1000 mg dose of intravenous paracetamol probably does not decrease pain as much as ultracaine PCB during procedure (median 2 points higher, IQR 1 to 3; P < 0.001; 1 RCT, 46 participants; moderate-certainty evidence). Various local anaesthetics in PCB comparative effectiveness trials A 10 mL buffered 2% lidocaine PCB probably does not result in a clinically meaningful difference in pain with dilation compared to a plain lidocaine PCB (MD -0.80, 95% CI -0.89 to -0.71; 1 RCT, 167 participants; moderate-certainty evidence). A buffered lidocaine PCB probably does not result in a clinically meaningful difference in pain with aspiration compared to plain lidocaine PCB (MD -0.57, 95% CI -1.01 to -0.06; 2 RCTs, 291 participants; moderate-certainty evidence). Non-PCB local anaesthesia or PCB technique effectiveness trials PCB: waiting versus no waiting Waiting three to five minutes between 1% lidocaine PCB injection and dilation probably does not result in a clinically meaningful difference in pain with dilation compared to not waiting (MD -0.70, 95% CI -1.23 to -0.17; 2 RCTs, 357 participants; moderate-certainty evidence). Topical cervical analgesia Topical 10 mL 2% lignocaine gel probably does not result in a clinically meaningful difference in pain with aspiration compared to KY Jelly (MD -0.87, 95% CI -1.60 to -0.14; 1 RCT, 131 participants; moderate-certainty evidence). In participants who also received a PCB, 20 mg topical cervical lidocaine spray probably does not result in a clinically meaningful difference in pain during the procedure compared to two pumps of normal saline spray (median -1 point, IQR -2 to -1; P < 0.001; 1 RCT, 55 participants; moderate-certainty evidence). Intravenous paracetamol 1000 mg compared to two pumps of cervical lidocaine spray probably does not results in a clinically meaningful difference in pain procedure (median 1 point, IQR -2 to 2; P < 0.001; 1 RCT, 48 participants; low-certainty evidence). Non-PCB local anaesthesia or PCB technique comparative effectiveness trials Depth of PCB The evidence suggests that a 3-cm deep PCB probably does not result in a clinically meaningful difference in pain with aspiration compared to a 1.5-cm deep PCB (MD -1.00, 95% CI -1.09 to -0.91; 2 RCTs, 229 participants; low-certainty evidence). PCB: four sites versus two sites A two-site (4-8 o'clock) 20 mL 1% lidocaine PCB does not result in a clinically meaningful difference in pain with dilation compared to a four-site (2-4-8-10 o'clock) PCB (MD 8.60, 95% CI 0.69 to 16.51; 1 RCT, 163 participants; high-certainty evidence). Overall, participants reported moderately high satisfaction with pain control and studies reported few adverse events. AUTHORS' CONCLUSIONS: Evidence from this updated review indicates that a 20 mL 1% plain lidocaine PCB decreases pain during an abortion procedure. Evidence supports forgoing buffering lidocaine and a wait time between PCB injection and cervical dilation. A 1.5-cm deep injection as opposed to a 3-cm deep injection is sufficient. A two-site PCB injection as opposed to a four-site injection has similar effectiveness. Topical cervical anaesthesia (10 mL 2% lignocaine gel or 20 mg topical cervical lidocaine spray) as compared to placebo did not decrease pain based on moderate-certainty evidence, but then when compared to PCB, pain control was similar. Due to this inconsistency in evidence regarding the effectiveness of topical anaesthesia, its routine use is presently not supported. This review did not include studies of pain management with conscious sedation but, based on the results of our prior Cochrane review and the 2022 WHO guidelines, we recommend that the option of combination of pain management using conscious sedation plus PCB and non-steroidal anti-inflammatory drugs should be offered where conscious sedation is available as it further decreases pain.

First and Second-Trimester Surgical Abortion Providers and Services in 2019: Results From the Canadian Abortion Provider Survey.

OBJECTIVE: Our objective was to explore the workforce and clinical care of first and second-trimester surgical abortion (FTSA, STSA) providers following the publication of the updated Society of Obstetricians and Gynaecologists of Canada (SOGC) surgical abortion guidelines. METHODS: We conducted a national, cross-sectional, online, self-administered survey of physicians who provided abortion care in 2019. This anonymized survey collected participant demographics, types of abortion services, and characteristics of FTSA and STSA clinical care. Through healthcare organizations using a modified Dillman technique, we recruited from July to December 2020. Descriptive statistics were generated by R Statistical Software. RESULTS: We present the data of 222 surgical abortion provider respondents, of whom 219 provided FTSA, 109 STSA, and 106 both. Respondents practiced in every Canadian province and territory. Most were obstetrician-gynaecologists (56.8%) and family physicians (36.0%). The majority of FTSA and STSA respondents were located in urban settings, 64.8% and 79.8%, respectively, and more than 80% practiced in hospitals. More than 1 in 4 respondents reported <5 years' experience with surgical abortion care and 93.2% followed SOGC guidelines. Noted guideline deviations included that prophylactic antibiotic use was not universal, and more than half of respondents used sharp curettage in addition to suction. Fewer than 5% of STSA respondents used mifepristone for cervical preparation. CONCLUSION: The surgical abortion workforce is multidisciplinary and rejuvenating. Education, training, and practice support, including SOGC guideline implementation, are required to optimize care and to ensure equitable FTSA and STSA access in both rural and urban regions. GESTATIONAL AGE NOTATION: weeks, weeks' gestation, gestational age (GA), e.g., 116 weeks.

Telemedicine for First-Trimester Medical Abortion in Canada: Results of a 2019 Survey.

Introduction: Telemedicine has the potential to improve abortion access disparities in Canada. We aimed to explore the provision of telemedicine for first-trimester medical abortion and related barriers in 2019. Methods: We conducted a national, cross-sectional, anonymized, web-based survey of clinicians who provided abortion care in 2019 in Canada. We distributed our survey through professional health organizations to maximize identification of possible eligible respondents and used a modified Dillman technique to foster responses. Questions elicited provider demographics, clinical characteristics, including telemedicine first-trimester medical abortion and perceived related barriers. Descriptive statistics were analyzed using R software. Results: Among 465 respondents, 388 reported providing first-trimester medical abortion across Canada; 44.0% reported experience using telemedicine for some components of care: 49.3% of primary care clinicians and 28.7% of specialists. Telemedicine was used for initial consultation (86.0%), prescription (82.2%), or follow-up (92.2%). The median percentage of telemedicine providers' patients who underwent a dating ultrasound was 90.0. The majority usually followed up with patients through quantitative human chorionic gonadotropin (hCG) (84.2%). Seventy-eight percent perceived barriers to telemedicine; the most common being inability to confirm gestational age with ultrasound (43.0%), and lack of provincial telemedicine abortion fee code to pay practitioners (30.2%), timely access to serum hCG testing (24.6%), and nearby emergency services (23.3%). Discussion: In 2019, fewer than half of respondents reported providing some aspects of first-trimester medical abortion through telemedicine and the majority perceived barriers. Our results can inform knowledge translation activities to reduce barriers and increase telemedicine abortion care in Canada.

Second- and Third-Trimester Medical Abortion Providers and Services in 2019: Results From the Canadian Abortion Provider Survey.

OBJECTIVES: Mifepristone became available in Canada in 2017. Updated national guidelines recommend its off-label use for second/third-trimester medical abortion (STMA/TTMA) by labour induction. The objective of this study was to explore STMA/TTMA provision in Canada and the role of mifepristone. METHODS: We conducted a national, cross-sectional, web-based, self-administered, anonymized survey, available in English and French. The survey was distributed through health professional organizations and recruited physicians who provided abortion care in 2019. We used a modified Dillman technique to maximize participation. The survey included sections on workforce and clinical care, including mifepristone use. We used R statistical software to produce descriptive statistics. RESULTS: Four hundred sixty-five clinicians responded to the survey, of whom 112 reported providing STMA and 63, TTMA, for a total of 115 respondents providing at least 1 of the 2 services. Two-thirds of respondents were general obstetrician-gynaecologists or family physicians and the remainder were maternal-fetal medicine subspecialists. The majority (64.7%) provided STMA/TTMA in an academic hospital, and 59.4% performed fewer than 5 STMAs (maximum 50) and 76.1%, fewer than 5 TTMA (maximum 15) in 2019. Fifty-nine percent of respondents reported having used mifepristone/misoprostol for STMA. Among mifepristone users, 48.6% used it for TTMA. Most required an indication beyond patient request to provide STMA/TTMA (82.1%/95.5%). CONCLUSIONS: STMA/TTMA care is provided by multiple (sub-) specialties, and mifepristone has not yet been universally implemented. Our results will inform knowledge translation activities aimed at facilitating collaboration between STMA/TTMA providers and health policy and service delivery leaders and will further increase mifepristone use for STMA/TTMA in Canada.

A discussion on the 'dispensable' amino acids.

PURPOSE OF REVIEW: Recently, the concept of dietary dispensable amino acids has been increasingly challenged, as more indirect and direct (experimental) evidence has pointed to the need for them in the diet during specific life stages or disease states. Here, we discuss the classification of amino acids, methods to assess the needs for dispensable amino acids with experimental evidence from our recent studies, and highlight the role of specific dispensable amino acids in metabolism and health. RECENT FINDINGS: There exist differences among the dispensable amino acids to act as effective nitrogen sources in humans. Glycine, a dispensable amino acid is conditionally indispensable in later stages of human pregnancy. SUMMARY: The so-called 'dispensable' amino acids are quantitatively nearly 75% of the daily protein needs in humans. In certain life-stages and diseases, there is a dietary demand for the dispensable amino acids. Future well-designed studies are required to identify the dietary demand for these amino acids, which will certainly be useful for dietary management in specific diseases and to maintain health across all life-stages.

Glycine, a Dispensable Amino Acid, Is Conditionally Indispensable in Late Stages of Human Pregnancy.

BACKGROUND: Recently, we showed that there are higher protein, lysine, and phenylalanine requirements in late stages of pregnancy compared with early stages. Animal studies have suggested an increased dietary need for specific dispensable amino acids in pregnancy; whether such a need exists in human pregnancies is unknown. OBJECTIVE: The objective of the current study was to examine whether healthy pregnant women at midgestation (20-29 wk) and late gestation (30-40 wk) have a dietary demand for glycine, a dispensable amino acid, using the indicator amino acid oxidation method and measurement of plasma 5-oxoproline concentrations. METHODS: Seventeen healthy women (aged 26-36 y) randomly received different test glycine intakes (range: 5-100 mg·kg-1·d-1) during each study day in midgestation (∼26 wk, n = 17 observations in 9 women) and late gestation (∼35 wk, n = 19 observations in 8 women). Diets were isocaloric with energy at 1.7 × resting energy expenditure. Protein was given as a crystalline amino acid mixture based on egg protein composition at current estimated average requirement (EAR; 0.88 g·kg-1·d-1). Breath samples were collected at baseline and isotopic steady state to measure oxidation of L-[1-13C]phenylalanine to 13CO2 (F13CO2). Plasma was collected at the sixth hour of the study day. Linear regression crossover analysis and simple linear regression were used to assess responses in F13CO2 and plasma 5-oxoproline concentrations to different glycine intakes. RESULTS: No statistically significant responses were observed in midgestation. However, in late gestation, lower glycine intakes resulted in higher rates of F13CO2 (suggesting low protein synthesis) with a breakpoint for phenylalanine oxidation at >37 mg glycine·kg-1·d-1 and higher plasma 5-oxoproline (suggesting low glycine availability) with a breakpoint >27 mg glycine·kg-1·d-1. CONCLUSIONS: The findings suggest that glycine should be considered a "conditionally" indispensable amino acid during late gestation, especially when protein intakes are at 0.88 g·kg-1·d-1, the current EAR. This trial was registered at clinicaltrials.gov as NCT02149953.

Histamine receptors and COVID-19.

OBJECTIVE: Reports that the over-the-counter histamine H2 receptor antagonist famotidine could help treat the novel coronavirus disease (COVID-19) appeared from April 2020. We, therefore, examined reports on interactions between severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and histamine receptor antagonists. METHODS: A systematic literature search was performed by 19 September 2020, and updated on 28 October 2020, in PubMed, Scopus, Cochrane Library and Google Scholar using (COVID-19 OR coronavirus OR SARS-CoV-2) AND (histamine antagonist OR famotidine OR cimetidine). ClinicalTrials.gov was searched for COVID-19 and (famotidine or histamine). RESULTS: Famotidine may be a useful addition in COVID-19 treatment, but the results from prospective randomized trials are as yet awaited. Bioinformatics/drug repurposing studies indicated that, among several medicines, H1 and H2 receptor antagonists may interact with key viral enzymes. However, in vitro studies have to date failed to show a direct inhibition of famotidine on SARS-CoV-2 replication. CONCLUSIONS: Clinical research into the potential benefits of H2 receptor antagonists in managing COVID-19 inflammation began from a simple observation and now is being tested in multi-centre clinical trials. The positive effects of famotidine may be due to H2 receptor-mediated immunomodulatory actions on mast cell histamine-cytokine cross-talk, rather than a direct action on SARS-CoV-2.

The perspective of Canadian health care professionals on abortion service during the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic and pandemic response created novel challenges for abortion services. Canada was uniquely positioned to transition to telemedicine because internationally common restrictions on abortion medication were removed before the pandemic. OBJECTIVE: We sought to characterize the experiences of abortion health care professionals in Canada during the COVID-19 pandemic and the impact of the pandemic response on abortion services. METHODS: We conducted a sequential mixed methods study between July 2020 and January 2021. We invited physicians, nurse practitioners and administrators to participate in a cross-sectional survey containing an open-ended question about the impact of the pandemic response on abortion care. We employed an inductive codebook thematic analysis, which informed the development of a second, primarily quantitative survey. RESULTS: Our initial survey had 307 respondents and our second had 78. Fifty-three percent were family physicians. Our first survey found respondents considered abortion access essential. We identified three key topicss: access to abortion care was often maintained despite pandemic-related challenges (e.g. difficulty obtaining tests, additional costs); change of practice to low-touch medication abortion care and provider perceptions of patient experience, including shifting demand, telemedicine acceptability and increased rural access. The second survey indicated uptake of telemedicine medication abortion among 89% of participants except in Quebec, where regulations meant procedures were nearly exclusively surgical. Restrictions did not delay care according to 76% of participants. CONCLUSIONS: Canadian health care professionals report their facilities deemed abortion an essential service. Provinces and territories, except Quebec, described a robust pandemic transition to telemedicine to ensure access to services. PODCAST: An accompanying podcast is available in the Supplementary Data, in which the authors Dr Madeleine Ennis and Kate Wahl discuss their research on how family planning care and access to abortion services have changed during the COVID-19 pandemic.

Access to abortion care was challenged by the response to COVID-19. Canada had fewer restrictions on medical abortion than many other countries when the pandemic began. The goal of this study was to describe the experiences of health care practitioners providing abortion in Canada and the impact of the pandemic and the pandemic response measures on abortion services. We conducted two surveys of physicians, nurse practitioners and administrators between July 2020 and January 2021. Most of the health care practitioners who participated reported that medical and surgical abortion care were essential and that, except in the province of Quebec, there was a rapid transition to virtual telemedicine care for first trimester abortions. Several practitioners said that virtual care made abortion more accessible. Other practitioners reported that it was challenging to order certain tests, access operating room facilities or make referrals for late second trimester cases. Practitioners felt that patients had strong fears about COVID-19 exposure and reported that limited contraception access was increasingly a reason for seeking abortion care. The results of the study suggested that abortion was considered essential and that the pandemic instigated a transition to virtual care in all provinces and territories except Quebec.

Dietary Aromatic Amino Acid Requirements During Early and Late Gestation in Healthy Pregnant Women.

BACKGROUND: Phenylalanine and tyrosine (referred to as total aromatic amino acids; TAAs) are essential for protein synthesis, and are precursors for important catecholamines. Current estimated average requirement (EAR) recommendations for TAA during pregnancy are 36 mg·kg-1·d-1, and has not been experimentally determined. OBJECTIVES: The aim was to determine TAA requirements (dietary phenylalanine in the absence of tyrosine) during early and late gestation using the indicator amino acid oxidation (IAAO, with L-[1-13C]leucine) technique. METHODS: Nineteen healthy pregnant women (age 22-38 y) were studied at a range of phenylalanine intakes (5 to 100 mg·kg-1·d-1) in early (13-19 wk) and/or late (33-39 wk) pregnancy for a total of 51 study days. Graded test intakes were provided as 8 hourly isonitrogenous and isocaloric meals. Breath samples were collected for 13C enrichment analysis on an isotope ratio mass spectrometer. A plasma sample was collected and analyzed for phenylalanine and tyrosine concentrations on an amino acid analyzer. The TAA requirement in early and late pregnancy was calculated using 2-phase linear regression crossover analysis that identified breakpoints in 13CO2 production (the requirement) in response to phenylalanine intakes. RESULTS: TAA requirement during early pregnancy was 44 mg·kg-1·d-1 (95% CI: 28.3, 58.8) and during late pregnancy was 50 mg·kg-1·d-1 (95% CI: 36.1, 63.1). In early and late pregnancy, plasma phenylalanine and tyrosine concentrations rose linearly in response to graded phenylalanine intakes. CONCLUSIONS: Our results suggest that the current EAR of 36 mg·kg-1·d-1 for TAAs is underestimated. When compared with results previously determined in nonpregnant adults, early pregnancy requirements were similar (43 compared with 44 mg·kg-1·d-1, respectively). During late pregnancy, a 14% higher TAA requirement was observed when compared with early pregnancy. The results from this study have potential implications for creating gestation stage-specific TAA recommendations.

Impact of enteral arginine supplementation on lysine metabolism in humans: A proof-of-concept for lysine-related inborn errors of metabolism.

Patients with lysine-related inborn errors of metabolism (pyridoxine-dependent epilepsy [PDE] and glutaric aciduria type 1 [GA1]), follow a lysine-restricted diet with arginine-fortified lysine-free amino acid formula and additional oral arginine supplementation as a newer therapy for PDE. The rationale of arginine supplementation is based on arginine's ability to compete with lysine transport across cell membranes via shared transporter systems. Adequate doses of arginine required to competitively inhibit enteral lysine uptake has not been studied in humans This proof-of-concept study investigates the effect of incremental enteral arginine doses on whole-body lysine oxidation using an in vivo stable isotope tracer, L-[1-13 C] lysine, in healthy humans. Five healthy men completed six study days each consuming one dose of l-arginine HCl per study day; range = 50-600 mg/kg/d. Lysine intake was at DRI (30 mg/kg/d). Breath samples were analysed for L-[1-13 C] lysine oxidation to 13 CO2 using an isotope ratio mass spectrometer. Plasma amino acid concentrations were analysed using an amino acid analyser. Increasing doses of l-arginine HCl caused a linear decrease in whole-body lysine oxidation. Plasma arginine concentration increased, and plasma lysine concentration decreased below normal range with high arginine intakes. We provide the first empirical evidence of arginine-lysine antagonism in response to increasing oral arginine doses. Results suggest 300-600 mg/kg/d of l-arginine HCl and lysine intake restricted to DRI is needed to reduce enteral lysine uptake and systemic lysine oxidation. This could potentially lead to a recommended dose for arginine in lysine-related inborn errors of metabolism.

Connectivity mapping (ssCMap) to predict A20-inducing drugs and their antiinflammatory action in cystic fibrosis.

Cystic fibrosis (CF) lung disease is characterized by chronic and exaggerated inflammation in the airways. Despite recent developments to therapeutically overcome the underlying functional defect in the cystic fibrosis transmembrane conductance regulator, there is still an unmet need to also normalize the inflammatory response. The prolonged and heightened inflammatory response in CF is, in part, mediated by a lack of intrinsic down-regulation of the proinflammatory NF-κB pathway. We have previously identified reduced expression of the NF-κB down-regulator A20 in CF as a key target to normalize the inflammatory response. Here, we have used publicly available gene array expression data together with a statistically significant connections' map (sscMap) to successfully predict drugs already licensed for the use in humans to induce A20 mRNA and protein expression and thereby reduce inflammation. The effect of the predicted drugs on A20 and NF-κB(p65) expression (mRNA) as well as proinflammatory cytokine release (IL-8) in the presence and absence of bacterial LPS was shown in bronchial epithelial cells lines (16HBE14o-, CFBE41o-) and in primary nasal epithelial cells from patients with CF (Phe508del homozygous) and non-CF controls. Additionally, the specificity of the drug action on A20 was confirmed using cell lines with tnfαip3 (A20) knockdown (siRNA). We also show that the A20-inducing effect of ikarugamycin and quercetin is lower in CF-derived airway epithelial cells than in non-CF cells.

Lung clearance index is a repeatable and sensitive indicator of radiological changes in bronchiectasis.

RATIONALE: In bronchiectasis there is a need for improved markers of lung function to determine disease severity and response to therapy. OBJECTIVES: To assess whether the lung clearance index is a repeatable and more sensitive indicator of computed tomography (CT) scan abnormalities than spirometry in bronchiectasis. METHODS: Thirty patients with stable bronchiectasis were recruited and lung clearance index, spirometry, and health-related quality of life measures were assessed on two occasions, 2 weeks apart when stable (study 1). A separate group of 60 patients with stable bronchiectasis was studied on a single visit with the same measurements and a CT scan (study 2). MEASUREMENTS AND MAIN RESULTS: In study 1, the intervisit intraclass correlation coefficient for the lung clearance index was 0.94 (95% confidence interval, 0.89 to 0.97; P < 0.001). In study 2, the mean age was 62 (10) years, FEV1 76.5% predicted (18.9), lung clearance index 9.1 (2.0), and total CT score 14.1 (10.2)%. The lung clearance index was abnormal in 53 of 60 patients (88%) and FEV1 was abnormal in 37 of 60 patients (62%). FEV1 negatively correlated with the lung clearance index (r = -0.51, P < 0.0001). Across CT scores, there was a relationship with the lung clearance index, with little evidence of an effect of FEV1. There were no significant associations between the lung clearance index or FEV1 and health-related quality of life. CONCLUSIONS: The lung clearance index is repeatable and a more sensitive measure than FEV1 in the detection of abnormalities demonstrated on CT scan. The lung clearance index has the potential to be a useful clinical and research tool in patients with bronchiectasis.

Increased expression of bronchial epithelial transient receptor potential vanilloid 1 channels in patients with severe asthma.

BACKGROUND: The airway epithelium is exposed to a range of physical and chemical irritants in the environment that are known to trigger asthma. Transient receptor potential (TRP) cation channels play a central role in sensory responses to noxious physical and chemical stimuli. Recent genetic evidence suggests an involvement of transient receptor potential vanilloid 1 (TRPV1), one member of the vanilloid subfamily of TRP channels, in the pathophysiology of asthma. The functional expression of TRPV1 on airway epithelium has yet to be elucidated. OBJECTIVE: In this study we examined the molecular, functional, and immunohistochemical expression of TRPV1 in asthmatic and healthy airways. METHODS: Bronchial biopsy specimens and bronchial brushings were obtained from healthy volunteers (n = 18), patients with mild-to-moderate asthma (n = 24), and patients with refractory asthma (n = 22). Cultured primary bronchial epithelial cells from patients with mild asthma (n = 4), nonasthmatic coughers (n = 4), and healthy subjects (n = 4) were studied to investigate the functional role of TRPV1. RESULTS: Quantitative immunohistochemistry revealed significantly more TRPV1 expression in asthmatic patients compared with healthy subjects, with the greatest expression in patients with refractory asthma (P = .001). PCR and Western blotting analysis confirmed gene and protein expression of TRPV1 in cultured primary bronchial epithelial cells. Patch-clamp electrophysiology directly confirmed functional TRPV1 expression in all 3 groups. In functional assays the TRPV1 agonist capsaicin induced dose-dependent IL-8 release, which could be blocked by the antagonist capsazepine. Reduction of external pH from 7.4 to 6.4 activated a capsazepine-sensitive outwardly rectifying membrane current. CONCLUSIONS: Functional TRPV1 channels are present in the human airway epithelium and overexpressed in the airways of patients with refractory asthma. These channels might represent a novel therapeutic target for the treatment of uncontrolled asthma.

Cystic Fibrosis from Laboratory to Bedside: The Role of A20 in NF-κB-Mediated Inflammation.

Cystic fibrosis (CF) is a lifelong, inflammatory multi-organ disease and the most common lethal, genetic condition in Caucasian populations, with a median survival rate of 41.5 years. Pulmonary disease, characterized by infective exacerbations, bronchiectasis and increasing airway insufficiency is the most serious manifestation of this disease process, currently responsible for over 80% of CF deaths. Chronic dysregulation of the innate immune and host inflammatory response has been proposed as a mechanism central to this genetic condition, primarily driven by the nuclear factor κB (NF-κB) pathway. Chronic activation of this transcription factor complex leads to the production of pro-inflammatory cytokines and mediators such as IL-6, IL-8 and TNF-α. A20 has been described as a central and inducible negative regulator of NF-κB. This intracellular molecule negatively regulates NF-κB-driven pro-inflammatory signalling upon toll-like receptor activation at the level of TRAF6 activation. Silencing of A20 increases cellular levels of p65 and induces a pro-inflammatory state. We have previously shown that A20 expression positively correlates with lung function (FEV1%) in CF. Despite improvement in survival rates in recent years, advancements in available therapies have been incremental. We demonstrate that the experimental use of naturally occurring plant diterpenes such as gibberellin on lipopolysaccharide-stimulated cell lines reduces IL-8 release in an A20-dependent manner. We discuss how the use of a novel bio-informatics gene expression connectivity-mapping technique to identify small molecule compounds that similarly mimic the action of A20 may lead to the development of new therapeutic approaches capable of reducing chronic airway inflammation in CF.

Lung microbiota and bacterial abundance in patients with bronchiectasis when clinically stable and during exacerbation.

RATIONALE: Characterization of bacterial populations in infectious respiratory diseases will provide improved understanding of the relationship between the lung microbiota, disease pathogenesis, and treatment outcomes. OBJECTIVES: To comprehensively define lung microbiota composition during stable disease and exacerbation in patients with bronchiectasis. METHODS: Sputum was collected from patients when clinically stable and before and after completion of antibiotic treatment of exacerbations. Bacterial abundance and community composition were analyzed using anaerobic culture and 16S rDNA pyrosequencing. MEASUREMENTS AND MAIN RESULTS: In clinically stable patients, aerobic and anaerobic bacteria were detected in 40 of 40 (100%) and 33 of 40 (83%) sputum samples, respectively. The dominant organisms cultured were Pseudomonas aeruginosa (n = 10 patients), Haemophilus influenzae (n = 12), Prevotella (n = 18), and Veillonella (n = 13). Pyrosequencing generated more than 150,000 sequences, representing 113 distinct microbial taxa; the majority of observed community richness resulted from taxa present in low abundance with similar patterns of phyla distribution in clinically stable patients and patients at the onset of exacerbation. After treatment of exacerbation, there was no change in total (P = 0.925), aerobic (P = 0.917), or anaerobic (P = 0.683) load and only a limited shift in community composition. Agreement for detection of bacteria by culture and pyrosequencing was good for aerobic bacteria such as P. aeruginosa (κ = 0.84) but poorer for other genera including anaerobes. Lack of agreement was largely due to bacteria being detected by pyrosequencing but not by culture. CONCLUSIONS: A complex microbiota is present in the lungs of patients with bronchiectasis and remains stable through treatment of exacerbations, suggesting that changes in microbiota composition do not account for exacerbations.

Inflammatory and cytotoxic effects of acrolein, nicotine, acetylaldehyde and cigarette smoke extract on human nasal epithelial cells.

BACKGROUND: Cigarette smoke induces a pro-inflammatory response in airway epithelial cells but it is not clear which of the various chemicals contained within cigarette smoke (CS) should be regarded as predominantly responsible for these effects. We hypothesised that acrolein, nicotine and acetylaldehyde, important chemicals contained within volatile cigarette smoke in terms of inducing inflammation and causing addiction, have immunomodulatory effects in primary nasal epithelial cell cultures (PNECs). METHODS: PNECs from 19 healthy subjects were grown in submerged cultures and were incubated with acrolein, nicotine or acetylaldehyde prior to stimulation with Pseudomonas aeruginosa lipopolysaccharide (PA LPS). Experiments were repeated using cigarette smoke extract (CSE) for comparison. IL-8 was measured by ELISA, activation of NF-κB by ELISA and Western blotting, and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labeling (TUNEL) method. RESULTS: CSE was pro-inflammatory after a 24 h exposure and 42% of cells were apoptotic or necrotic after this exposure time. Acrolein was pro-inflammatory for the PNEC cultures (30 µM exposure for 4 h inducing a 2.0 fold increase in IL-8 release) and also increased IL-8 release after stimulation with PA LPS. In contrast, nicotine had anti-inflammatory properties (0.6 fold IL-8 release after 50 µM exposure to nicotine for 24 h), and acetylaldehyde was without effect. Acrolein and nicotine had cellular stimulatory and anti-inflammatory effects respectively, as determined by NF-κB activation. Both chemicals increased levels of cleaved caspase 3 and induced cell death. CONCLUSIONS: Acrolein is pro-inflammatory and nicotine anti-inflammatory in PNEC cultures. CSE induces cell death predominantly by apoptotic mechanisms.

Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells.

The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF.

Airway epithelial cell apoptosis and inflammation in COPD, smokers and nonsmokers.

We hypothesised that primary bronchial epithelial cells (PBECs) from subjects with chronic obstructive pulmonary disease (COPD) respond differently to Pseudomonas aeruginosa lipopolysaccharide (LPS) after cigarette smoke extract (CSE) exposure than PBECs obtained from smokers without airflow obstruction and nonsmokers. PBECs from 16 COPD subjects, 10 smokers without airflow obstruction and nine nonsmokers were cultured at air-liquid interface. Cultures were incubated with CSE prior to stimulation with P. aeruginosa LPS. Interleukin (IL)-6 and IL-8 were measured by ELISA and Toll-like receptor (TLR)-4 expression by fluorescence-activated cell sorter. Activation of nuclear factor (NF)-κB was determined by Western blotting and ELISA, and MAPK and caspase-3 activity by Western blotting. Apoptosis was evaluated using Annexin-V staining and the terminal transferase-mediated dUTP nick end-labelling methods. Constitutive release of IL-8 and IL-6 was greatest from the COPD cultures. However, CSE pretreatment followed by P. aeruginosa LPS stimulation reduced IL-8 release from COPD PBECs, but increased it from cells of smokers without airflow obstruction and nonsmokers. TLR-4 expression, MAPK and NF-κB activation in COPD cultures were reduced after CSE treatment, but not in the smokers without airflow obstruction or nonsmoker groups, which was associated with increased apoptosis. CSE attenuates inflammatory responses to LPS in cells from people with COPD but not those from nonsmoking individuals and those who smoke without airflow obstruction.

Expression of the nuclear factor-κB inhibitor A20 is altered in the cystic fibrosis epithelium.

A20 is a lipopolysaccharide (LPS)-inducible, cytoplasmic zinc finger protein, which inhibits Toll-like receptor-activated nuclear factor (NF)-κB signalling by deubiquitinating tumour necrosis factor receptor-associated factor (TRAF)-6. The action of A20 is facilitated by complex formation with ring finger protein (RNF)-11, Itch and TAX-1 binding protein-1 (TAX1BP1). This study investigated whether the expression of A20 is altered in the chronically inflamed cystic fibrosis (CF) airway epithelium. Nasal epithelial cells from CF patients (F508del homozygous), non-CF controls and immortalised epithelial cells (16HBE14o- and CFBE41o-) were stimulated with LPS. Cytoplasmic expression of A20 and expression of NF-κB subunits were analysed. Formation of the A20 ubiquitin editing complex was also investigated. In CFBE41o-, peak LPS-induced A20 expression was delayed compared with 16HBE14o- and fell significantly below basal levels 12-24 h after LPS stimulation. This was confirmed in primary CF airway cells. Additionally, a significant inverse relationship between A20 and p65 expression was observed. Inhibitor studies showed that A20 does not undergo proteasomal degradation in CFBE41o-. A20 interacted with TAX1BP1, RNF11 and TRAF6 in 16HBE14o- cells, but these interactions were not observed in CFBE41o-. The expression of A20 is significantly altered in CF, and important interactions with complex members and target proteins are lost, which may contribute to the state of chronic NF-κB-driven inflammation.

Development and pilot testing of the 2019 Canadian Abortion Provider Survey.

BACKGROUND: Substantial changes in abortion care regulations, available medications and national clinical practice guidelines have occurred since a 2012 national Canadian Abortion Provider Survey (CAPS). We developed and piloted the CAPS 2019 survey instrument to explore changes of the abortion provider workforce, their clinical care as well as experiences with stigma and harassment. METHODS: We undertook development and piloting in three phases: (1) development of the preliminary survey sections and questions based on the 2012 survey instrument, (2) content validation and feasibility of including certain content aspects via a modified Delphi Method with panels of clinical and research experts, and (3) pilot testing of the draft survey for face validity and clarity of language; assessing usability of the web-based Research Electronic Data Capture platform including the feasibility of complex skip pattern functionality. We performed content analysis of phase 2 results and used a general inductive approach to identify necessary survey modifications. RESULTS: In phase 1, we generated a survey draft that reflected the changes in Canadian abortion care regulations and guidelines and included questions for clinicians and administrators providing first and second trimester surgical and medical abortion. In phase 2, we held 6 expert panel meetings of 5-8 participants each representing clinicians, administrators and researchers to provide feedback on the initial survey draft. Due to the complexity of certain identified aspects, such as interdisciplinary collaboration and interprovincial care delivery differences, we revised the survey sections through an iterative process of meetings and revisions until we reached consensus on constructs and questions to include versus exclude for not being feasible. In phase 3, we made minor revisions based on pilot testing of the bilingual, web-based survey among additional experts chosen to be widely representative of the study population. Demonstrating its feasibility, we included complex branching and skip pattern logic so each respondent only viewed applicable questions based on their prior responses. CONCLUSIONS: We developed and piloted the CAPS 2019 survey instrument suitable to explore characteristics of a complex multidisciplinary workforce, their care and experience with stigma on a national level, and that can be adapted to other countries.