Time trends in diagnostic testing for primary ciliary dyskinesia in Europe

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Frequent mutation of histone-modifying genes in non-Hodgkin lymphoma

Follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) are the two most common non-Hodgkin lymphomas (NHLs). Here we sequenced tumour and matched normal DNA from 13 DLBCL cases and one FL case to identify genes with mutations in B-cell NHL. We analysed RNA-seq data from these and another 113 NHLs to identify genes with candidate mutations, and then re-sequenced tumour and matched normal DNA from these cases to confirm 109 genes with multiple somatic mutations. Genes with roles in histone modification were frequent targets of somatic mutation. For example, 32% of DLBCL and 89% of FL cases had somatic mutations in MLL2, which encodes a histone methyltransferase, and 11.4% and 13.4% of DLBCL and FL cases, respectively, had mutations in MEF2B, a calcium-regulated gene that cooperates with CREBBP and EP300 in acetylating histones. Our analysis suggests a previously unappreciated disruption of chromatin biology in lymphomagenesis

The NeST (Neoadjuvant systemic therapy in breast cancer) study: National Practice Questionnaire of United Kingdom multi-disciplinary decision making

Abstract: Background: Neoadjuvant systemic therapy (NST) is increasingly used in the treatment of breast cancer, yet it is clear that there is significant geographical variation in its use in the UK. This study aimed to examine stated practice across UK breast units, in terms of indications for use, radiological monitoring, pathological reporting of treatment response, and post-treatment surgical management. Methods: Multidisciplinary teams (MDTs) from all UK breast units were invited to participate in the NeST study. A detailed questionnaire assessing current stated practice was distributed to all participating units in December 2017 and data collated securely usingREDCap. Descriptive statistics were calculated for each questionnaire item. Results: Thirty-nine MDTs from a diverse range of hospitals responded. All MDTs routinely offered neoadjuvant chemotherapy (NACT) to a median of 10% (range 5–60%) of patients. Neoadjuvant endocrine therapy (NET) was offered to a median of 4% (range 0–25%) of patients by 66% of MDTs. The principal indication given for use of neoadjuvant therapy was for surgical downstaging. There was no consensus on methods of radiological monitoring of response, and a wide variety of pathological reporting systems were used to assess tumour response. Twenty-five percent of centres reported resecting the original tumour footprint, irrespective of clinical/radiological response. Radiologically negative axillae at diagnosis routinely had post-NACT or post-NET sentinel lymph node biopsy (SLNB) in 73.0 and 84% of centres respectively, whereas 16% performed SLNB pre-NACT. Positive axillae at diagnosis would receive axillary node clearance at 60% of centres, regardless of response to NACT. Discussion: There is wide variation in the stated use of neoadjuvant systemic therapy across the UK, with general low usage of NET. Surgical downstaging remains the most common indication of the use of NAC, although not all centres leverage the benefits of NAC for de-escalating surgery to the breast and/or axilla. There is a need for agreed multidisciplinary guidance for optimising selection and management of patients for NST. These findings will be corroborated in phase II of the NeST study which is a national collaborative prospective audit of NST utilisation and clinical outcomes

Rehabilitation versus surgical reconstruction for non-acute anterior cruciate ligament injury (ACL SNNAP): a pragmatic randomised controlled trial

BackgroundAnterior cruciate ligament (ACL) rupture is a common debilitating injury that can cause instability of the knee. We aimed to investigate the best management strategy between reconstructive surgery and non-surgical treatment for patients with a non-acute ACL injury and persistent symptoms of instability.MethodsWe did a pragmatic, multicentre, superiority, randomised controlled trial in 29 secondary care National Health Service orthopaedic units in the UK. Patients with symptomatic knee problems (instability) consistent with an ACL injury were eligible. We excluded patients with meniscal pathology with characteristics that indicate immediate surgery. Patients were randomly assigned (1:1) by computer to either surgery (reconstruction) or rehabilitation (physiotherapy but with subsequent reconstruction permitted if instability persisted after treatment), stratified by site and baseline Knee Injury and Osteoarthritis Outcome Score—4 domain version (KOOS4). This management design represented normal practice. The primary outcome was KOOS4 at 18 months after randomisation. The principal analyses were intention-to-treat based, with KOOS4 results analysed using linear regression. This trial is registered with ISRCTN, ISRCTN10110685, and ClinicalTrials.gov, NCT02980367.FindingsBetween Feb 1, 2017, and April 12, 2020, we recruited 316 patients. 156 (49%) participants were randomly assigned to the surgical reconstruction group and 160 (51%) to the rehabilitation group. Mean KOOS4 at 18 months was 73·0 (SD 18·3) in the surgical group and 64·6 (21·6) in the rehabilitation group. The adjusted mean difference was 7·9 (95% CI 2·5–13·2; p=0·0053) in favour of surgical management. 65 (41%) of 160 patients allocated to rehabilitation underwent subsequent surgery according to protocol within 18 months. 43 (28%) of 156 patients allocated to surgery did not receive their allocated treatment. We found no differences between groups in the proportion of intervention-related complications.InterpretationSurgical reconstruction as a management strategy for patients with non-acute ACL injury with persistent symptoms of instability was clinically superior and more cost-effective in comparison with rehabilitation management

KCa3.1 K+ Channel Expression and Function in Human Bronchial Epithelial Cells

The KCa3.1 K+ channel has been proposed as a novel target for pulmonary diseases such as asthma and pulmonary fibrosis. It is expressed in epithelia but its expression and function in primary human bronchial epithelial cells (HBECs) has not been described. Due to its proposed roles in the regulation of cell proliferation, migration, and epithelial fluid secretion, inhibiting this channel might have either beneficial or adverse effects on HBEC function. The aim of this study was to assess whether primary HBECs express the KCa3.1 channel and its role in HBEC function. Primary HBECs from the airways of healthy and asthmatic subjects, SV-transformed BEAS-2B cells and the neoplastic H292 epithelial cell line were studied. Primary HBECs, BEAS-2B and H292 cells expressed KCa3.1 mRNA and protein, and robust KCa3.1 ion currents. KCa3.1 protein expression was increased in asthmatic compared to healthy airway epithelium in situ, and KCa3.1 currents were larger in asthmatic compared to healthy HBECs cultured in vitro. Selective KCa3.1 blockers (TRAM-34, ICA-17043) had no effect on epithelial cell proliferation, wound closure, ciliary beat frequency, or mucus secretion. However, several features of TGFβ1-dependent epithelial-mesenchymal transition (EMT) were inhibited by KCa3.1 blockade. Treatment with KCa3.1 blockers is likely to be safe with respect to airway epithelial biology, and may potentially inhibit airway remodelling through the inhibition of EMT

Inhibition of the K_Ca3.1 channel attenuates features of TGFβ1-dependent EMT.

<p>(<b>A</b>) Cell elongation, quantified by a ratio of cell length:cell width of vimentin-stained BEAS-2B cells after 72 h, was increased by TGFβ1 or TGFβ1+DMSO compared to untreated cells (0.1% PBS/BSA; *P < 0.001). Pre-treatment of BEAS-2B cells with 200 nM TRAM-34 (#P < 0.001) or 100 nM ICA-17043 (##P = 0.027) significantly inhibited TGFβ1-induced cell elongation compared to TGFβ1+DMSO control. In contrast, TRAM-7, an inactive analog of TRAM-34, did not inhibit TGFβ1-induced cell elongation (**P = 0.022 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (<b>B</b>) BEAS-2B cells treated with 10 ng/ml TGFβ1 or TGFβ1+DMSO for 72 h exhibited a loss of E-cadherin expression in comparison to untreated cells (0.1% PBS/BSA; *P < 0.001, **P = 0.001). Pre-treatment with TRAM-34 (200 nM; #P = 0.013) or ICA-17043 (100 nM; ##P = 0.002) attenuated the TGFβ1-dependent loss of E-cadherin compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not prevent TGFβ1-induced loss of E-cadherin immunostaining (***P = 0.014 compared to TRAM-34). Data are presented as mean ± SEM from 6 individual experiments. (<b>C</b>) BEAS-2B cells treated with 10 ng/ml TGFβ1 or 10 ng/ml TGFβ1+DMSO control for 72 h displayed an increase in collagen-1 expression in comparison to untreated cells (0.1% PBS/BSA) (*P = 0.005; **P = 0.028 respectively). However, pre-treatment with TRAM-34 (200 nM; #P = 0.021) or ICA-17043 (100 nM; ##P = 0.024) significantly inhibited this effect compared to TGFβ1+DMSO. TRAM-7 (200 nM) did not inhibit TGFβ1-induced upregulation of collagen-1 immunostaining (***P = 0.021 compared to ICA-17043). Data are presented as mean ± SEM from 5 individual experiments.</p

Human bronchial epithelial cells express K_Ca3.1 mRNA and protein, and K_Ca3.1 expression is upregulated in the asthmatic bronchial epithelium.

<p><b>(A)</b> K_Ca3.1 mRNA (predicted PCR product size: 130 bp) was detected in monolayers of primary HBECs isolated from both patients with asthma (n = 10; denoted with “A”) and non-asthmatic healthy controls (n = 5; denoted with “NA”), alongside the housekeeping gene β-actin (predicted PCR product size: 146 bp). (<b>B</b>) qPCR revealed that K_Ca3.1 mRNA was expressed at similar levels in primary HBECs isolated from patients with asthma (n = 10) and healthy controls (n = 5). (<b>C</b>) An immunoreactive band of the appropriate size (K_Ca3.1: 48 kDa; β-actin: 42 kDa) was detected in lysates of primary HBECs isolated from two patients with asthma and one healthy control. (<b>D</b>) Quantification of K_Ca3.1 immunostaining by threshold analysis revealed that K_Ca3.1 expression was significantly elevated in asthmatic bronchial epithelium (*P = 0.007). (<b>E</b>) K_Ca3.1 immunostaining was significantly increased in severe asthma compared to mild asthma (**P = 0.008) and compared with healthy controls (*P = 0.002). (<b>F</b>) K_Ca3.1 and MUC5AC immunostaining co-localised in bronchial epithelial cells of sequential sections of biopsies from patients with asthma and healthy controls. (<b>G</b>) Quantification of MUC5AC immunostaining by threshold analysis revealed that MUC5AC expression was significantly increased in asthmatic bronchial epithelium (*P = 0.030), and (<b>H</b>) this was driven by a significant difference between the severe asthma and healthy control groups (*P = 0.034). (<b>I</b>) A significant correlation was found between K_Ca3.1 and MUC5AC immunostaining across the different severities of asthma and the healthy control groups (P < 0.001; r_s = 0.608). All data are plotted as median ± interquartile range; horizontal bars represent medians.</p

The K_Ca3.1 channel does not regulate epithelial mucus production or secretion.

<p>(<b>A</b>) Recombinant human amphiregulin (rh-AR) upregulated MUC5AC mRNA expression in H292 cells cultured in 6 well plates in a concentration-dependent manner after 24 h (*P = 0.010; **P = 0.028; n = 3 individual experiments). (<b>B</b>) Pre-treatment of H292 cells with the K_Ca3.1 blocker TRAM-34 (200 nM) for 30 min prior to stimulation with 10 ng/ml rh-AR for 24 h did not prevent rh-AR-induced MUC5AC mRNA expression (*P = 0.008, **P = 0.042; n = 6 individual experiments). rh-AR dose-dependently increased the mucin content of (<b>C</b>) H292 ALI culture lysates (*P = 0.029; n = 3 individual experiments) and (<b>D</b>) H292 ALI culture apical washes (*P = 0.009; n = 4 individual experiments) after 24 h, analysed by ELLA. Pre-treatment of H292 ALI cultures with 200 nM TRAM-34 did not prevent rh-AR-induced upregulation of mucin content within (<b>E</b>) H292 lysates (*P = 0.019; n = 6 individual experiments) or (<b>F</b>) apical washes (*P = 0.027; n = 3 individual experiments). Data are expressed as mean ± SEM.</p

The K_Ca3.1 channel does not regulate airway epithelial ciliary beat frequency.

<p>(<b>A</b>) Ciliary beat frequency of epithelial cells from healthy controls treated with either TRAM-34 or DMSO (n = 3). (<b>B</b>) Ciliary beat frequency of epithelial cells from asthmatic subjects treated with either TRAM-34 or DMSO (n = 3). *P = 0.001; data are expressed as mean ± SEM.</p

Asthmatic HBECs exhibit significantly larger K_Ca3.1 currents than healthy HBECs.

<p>(<b>A</b>) Current-voltage curves demonstrating that baseline whole cell membrane currents recorded from asthmatic and healthy primary HBECs were similar. (<b>B</b>) The 1-EBIO-dependent (1-EBIO minus baseline) currents recorded from asthmatic HBECs (n = 29 cells from 8 donors) were significantly larger than those from healthy controls (n = 16 cells from 5 donors); *P = 0.006 at +40 mV. (<b>C</b>) Raw 1-EBIO-dependent current trace demonstrating characteristic features of K_Ca3.1. The voltage protocol is shown inset. The K_Ca3.1 channel blocker TRAM-34 (200 nM) inhibited currents induced by 1-EBIO in both (<b>D</b>) asthmatic HBECs (n = 19 cells from 8 donors) and (<b>F</b>) healthy HBECs (n = 14 cells from 5 donors). Data are presented as mean ± SEM.</p