Feasibility of cardiopulmonary exercise testing in idiopathic pulmonary fibrosis

This is the author accepted manuscript. The final version is available from BMJ Publishing Group via the DOI in this recordBritish Thoracic Society Winter Meeting 2018, London, UK, 5-7 December 2018Introduction Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of irreversible declining lung function. Reductions in forced vital capacity (FVC) and diffusion capacity for carbon monoxide (DLCO) are the common clinical endpoints for prognostic monitoring and assessing treatment outcomes. The use of cardiopulmonary exercise testing (CPET) in IPF remains largely unexplored. Objectives To explore the feasibility of CPET as a clinical measure in IPF and identify associations with established clinical variables. Methods Seventeen patients with IPF were approached, and fifteen (88%) were recruited (13 male, 68.1±7.5 years). Incremental exercise testing to exhaustion was undertaken via electronically braked cycle ergometer. Variables included: peak oxygen consumption (VO2peak), peak work rate (WRpeak), nadir SpO2, ventilatory drive (VE/VCO2), alongside standard clinical pulmonary function tests of FVC and DLCO. Pearson’s correlation coefficients established relationships between variables. Results One participant was excluded (high baseline systolic blood pressure). Eight out of fourteen (57%) participants reached volitional exhaustion. Five CPETs were terminated early due to desaturation (SpO2 <88%) and one to an exercise-induced right bundle branch block (recovery within minutes of ceasing exercise). Mean (±SD) pulmonary and exercise results were: FVC, 84.9%±17.0%; DLCO, 56.5%±11.4%; VO2peak, 1.4±0.4 L.min-1, 16.5±5.5 mL.kg-1.min-1; WRpeak, 104±42 W; SpO2, 90±3%; VE/VCO2, 27.1±6.4. Significant correlations were identified between: FVC and SpO2 (r=0.58, p=0.032), DLCO and VE/VCO2 (r=0.81, p<0.001) and WRpeak (r=0.58, p=0.03). Body-mass relative VO2peak held moderate, but not significant relationships with FVC (r=0.44, p=0.11) and DLCO (r=0.53, p=0.51). Conclusions Initial findings from this study have found CPET to be acceptable to patients with IPF and potentially feasible as a testing measure. Preliminary results identified common exercise desaturation, suggesting less conservative SpO2 termination criteria (e.g. 80% cut-off) could be considered. Although exercise parameters held limited relationships with FVC and DLCO, results from VO2peak identifies potential additional and dynamic prognostic information and warrants further investigation.Royal Devon & Exeter Hospita

Cardiopulmonary Exercise Testing as a Longitudinal Clinical Tool in Interstitial Lung Disease Management

This is an abstract from International Conference of the American-Thoracic-Society Location: Dallas, TX Date: MAY 17-22, 2019Royal Devon & Exeter Hospita

NF-κB2 signalling in enteroids modulates enterocyte responses to secreted factors from bone marrow-derived dendritic cells

Alternative pathway NF-κB signalling regulates susceptibility towards developing inflammatory bowel disease (IBD), colitis-associated cancer and sepsis-associated intestinal epithelial cell apoptosis and shedding. However, the cell populations responsible for the perturbed alternative pathway NF-κB signalling in intestinal mucosal pathology remain unclear. In order to investigate the contribution of the epithelial compartment, we have tested whether NF-κB2 regulated transcription in intestinal epithelial cells controls the intestinal epithelial response to cytokines that are known to disrupt intestinal barrier permeability. Enteroids were generated from the proximal, middle and distal regions of small intestine (SI) from C57BL/6J wild-type mice and displayed region-specific morphology that was maintained during sub-culture. Enteroids treated with 100 ng/mL TNF were compared with corresponding regions of SI from C57BL/6J mice treated systemically with 0.33 mg/kg TNF for 1.5 h. TNF-induced apoptosis in all regions of the intestine in vitro and in vivo but resulted in Paneth cell degranulation only in proximal tissue-derived SI and enteroids. TNF also resulted in increased enteroid sphericity (quantified as circularity from two-dimensional bright field images). This response was dose and time-dependent and correlated with active caspase-3 immunopositivity. Proximal tissue-derived enteroids generated from Nfκb2−/− mice showed a significantly blunted circularity response following the addition of TNF, IFNγ, lipopolysaccharide (LPS) activated C57BL/6J-derived bone marrow-derived dendritic cells (BMDC) and secreted factors from LPS-activated BMDCs. However, Nfκb1−/− mouse-derived enteroids showed no significant changes in response to these stimuli. In conclusion, the selection of SI region is important when designing enteroid studies as region-specific identity and response to stimuli such as TNF are maintained in culture. Intestinal epithelial cells are at least partially responsible for regulating their own fate by modulating NF-κB2 signalling in response to stimuli known to be involved in multiple intestinal and systemic diseases. Future studies are warranted to investigate the therapeutic potential of intestinal epithelial NF-κB2 inhibition

Timing of Contact X-ray Brachytherapy in organ-preserving treatment of rectal cancer

Timing of Contact X-ray Brachytherapy in organ-preserving treatment of small rectal cancer Objective For patients with early rectal cancer, who are either at high risk for or refuse surgery, a planned organ preservation treatment involving a combination of external beam radiotherapy (EBRT) and Contact X-ray Brachytherapy (CXB) can be offered as an alternative option to surgery. (1-3) However, the ideal sequence of treatment for small rectal tumours (≤3cm), whether to administer CXB first or after EBRT, has not yet been well established, leading to variable sequences of this organ-preserving treatment being used.(3-5) This study has compared the oncological outcomes between the two treatment approaches using propensity score matching and inverse probability treatment weighting (IPTW) analysis to evaluate whether starting with CXB confers any benefits to patients. Method We analysed patients who had undergone both EBRT and CXB with curative intent, regardless of the treatment sequence, from the prospectively collected database at Clatterbridge Cancer Centre (2008-2019). Only patients who had well to moderately differentiated rectal adenocarcinoma (cT1-3, cN0-1, cM0) and small tumour size (≤ 3cm) were included. The variables of age, sex, fitness for surgery, performance status, tumour stage, nodal stage, EBRT regimen and CXB total dose, were considered possible confounders of the association between treatment regimen and outcomes. The balance of covariates before and after propensity matching and IPTW was assessed by examining the standardised mean difference (SMD) between the groups (Figure 1). The oncological outcomes based on the treatment sequence were first assessed in an unadjusted analysis followed by an adjusted model analysis considering all variables as confounders. Then, we performed propensity score matching (nearest-neighbour method, calliper= 0.25) and calculated IPTW to weigh the full cohort in each regression model. Statistical analysis was performed in R 4.3.1. The primary outcome measures were overall survival (OS) and disease-free survival (DFS). Secondary outcome measures consisted of the local regrowth rate, organ preservation rate, and presence of post-treatment rectal bleeding. Results A total of 251 eligible patients, who received either EBRT (n=103) or CXB (n=148) as their initial treatment with curative intent were included in the study. Patients received a CXB dose of 90-110Gy in 3-4 fractions over 4-6 weeks (each fraction two weeks apart) either before or after EBRT. EBRT was administered either as long-course chemoradiotherapy (45-50Gy/25 #/35 days), long-course radiotherapy alone (45Gy/20#/28days), or short-course radiotherapy (25Gy/5#/5 days). Following treatment, a watch-and-wait policy was adopted for patients who achieved a clinical complete/near response. The median follow-up was 37 [IQR:18-56] months for the EBRT-first group and 32 [IQR:16-54] months for the CXB-first group. In the unadjusted analysis, a higher risk of grade-1(26%) and grade-2(6%) rectal bleeding (p=0.008) was observed in patients who started with CXB, but no significant differences in any of the survival parameters were found. Analysis using the adjusted, propensity matching, and IPTW models, demonstrated a significant improvement of OS (p=0.04, HR (95%CI): 0.69 (0.48-0.98) and a higher risk of grade 1-2 rectal bleeding (p=0.01, OR (95%CI): 2.35(1.16-4.76) in those patients who had been received CXB as their initial treatment (Figure 2). However, DFS (p=0.87), local regrowth rate (p=0.70), and organ preservation rate (p=0.80) were not significantly different between the two groups. Conclusion Small rectal cancer (≤3cm), commencing treatment with CXB, as opposed to EBRT, was associated with improved overall survival, despite an increased risk of grade 1 and 2 rectal bleeding. However, there was no statistically significant improvement in terms of disease-free survival, local regrowth rate, or organ preservation rate with this treatment strategy

Mice lacking NF-κB1 exhibit marked DNA damage responses and more severe gastric pathology in response to intraperitoneal tamoxifen administration

Tamoxifen (TAM) has recently been shown to cause acute gastric atrophy and metaplasia in mice. We have previously demonstrated that the outcome of Helicobacter felis infection, which induces similar gastric lesions in mice, is altered by deletion of specific NF-κB subunits. Nfkb1-/- mice developed more severe gastric atrophy than wild-type (WT) mice 6 weeks after H. felis infection. In contrast, Nfkb2-/- mice were protected from this pathology. We therefore hypothesized that gastric lesions induced by TAM may be similarly regulated by signaling via NF-κB subunits. Groups of five female C57BL/6 (WT), Nfkb1-/-, Nfkb2-/- and c-Rel-/- mice were administered 150 mg/kg TAM by IP injection. Seventy-two hours later, gastric corpus tissues were taken for quantitative histological assessment. In addition, groups of six female WT and Nfkb1-/- mice were exposed to 12 Gy γ-irradiation. Gastric epithelial apoptosis was quantified 6 and 48 h after irradiation. TAM induced gastric epithelial lesions in all strains of mice, but this was more severe in Nfkb1-/- mice than in WT mice. Nfkb1-/- mice exhibited more severe parietal cell loss than WT mice, had increased gastric epithelial expression of Ki67 and had an exaggerated gastric epithelial DNA damage response as quantified by γH2AX. To investigate whether the difference in gastric epithelial DNA damage response of Nfkb1-/- mice was unique to TAM-induced DNA damage or a generic consequence of DNA damage, we also assessed gastric epithelial apoptosis following γ-irradiation. Six hours after γ-irradiation, gastric epithelial apoptosis was increased in the gastric corpus and antrum of Nfkb1-/- mice. NF-κB1-mediated signaling regulates the development of gastric mucosal pathology following TAM administration. This is associated with an exaggerated gastric epithelial DNA damage response. This aberrant response appears to reflect a more generic sensitization of the gastric mucosa of Nfkb1-/- mice to DNA damage

Validity and repeatability of cardiopulmonary exercise testing in interstitial lung disease

This is the final version. Available from BMC via the DOI in this record. Availability of data and materials: Data cannot be deposited in open access repositories for ethical reasons. Please contact the corresponding author (CAW) to discuss data access.Background: Cardiopulmonary exercise testing (CPET), and its primary outcome of peak oxygen uptake (VO2peak), are acknowledged as biomarkers in the diagnostic and prognostic management of interstitial lung disease (ILD). However, the validity and repeatability of CPET in those with ILD has yet to be fully characterised, and this study flls this evidence gap. Methods: Twenty-six people with ILD were recruited, and 21 successfully completed three CPETs. Of these, 17 completed two valid CPETs within a 3-month window, and 11 completed two valid CPETs within a 6-month window. Technical standards from the European Respiratory Society established validity, and repeatability was determined using mean change, intraclass correlation coefcient and typical error. Results: Every participant (100%) who successfully exercised to volitional exhaustion produced a maximal, and therefore valid, CPET. Approximately 20% of participants presented with a plateau in VO2, the primary criteria for establishing a maximal efort. The majority of participants otherwise presented with secondary criteria of respiratory exchange ratios in excess of 1.05, and maximal heart rates in excess of their predicted values. Repeatability analyses identifed that the typical error (expressed as percent of coefcient of variation) was 20% over 3-months in those reaching volitional exhaustion. Conclusion: This work has, for the frst time, fully characterised how patients with ILD respond to CPET in terms of primary and secondary verifcation criteria, and generated novel repeatability data that will prove useful in the assessment of disease progression, and future evaluation of therapeutic regimens where VO2peak is used as an outcome measure.Royal Devon and Exeter NHS Foundation Trust HospitalNational Institute for Health Research (NIHR)GW4 BioMed Medical Research Counci

The utility of the oxygen uptake efficiency plateau as a submaximal exercise biomarker in interstitial lung disease

This is the author accepted manuscript. The final version is available from BMJ Publishing Group via the DOI in this recordPaper S14 presented at the British Thoracic Society Winter Meeting, 17 - 19 February 202

The Nrf2 inhibitor brusatol is a potent antitumour agent in an orthotopic mouse model of colorectal cancer

© Evans et al. Nrf2 is a transcription factor that regulates cellular stress response and irinotecan-metabolising pathways. Its aberrant activity has been reported in a number of cancers, although relatively few studies have explored a role for Nrf2 in colorectal cancer (CRC). This study assessed the expression of Nrf2 in patient CRC tissues and explored the effect of Nrf2 modulation alone, or in combination with irinotecan, in human (HCT116) and murine (CT26) cell lines in vitro and in an orthotopic syngeneic mouse model utilising bioluminescent imaging. Using a tissue microarray, Nrf2 was found to be overexpressed (p < 0.01) in primary CRC and metastatic tissue relative to normal colon, with a positive correlation between Nrf2 expression in matched primary and metastatic samples. In vitro experiments in CRC cell lines revealed that Nrf2 siRNA and brusatol, which is known to inhibit Nrf2, decreased viability and sensitised cells to irinotecan toxicity. Furthermore, brusatol effectively abrogated CRC tumour growth in subcutaneously and orthotopicallyallografted mice, resulting in an average 8-fold reduction in luminescence at the study end-point (p=0.02). Our results highlight Nrf2 as a promising drug target in the treatment of CRC

An Open-Format Enteroid Culture System for Interrogation of Interactions Between Toxoplasma gondii and the Intestinal Epithelium

When transmitted through the oral route, Toxoplasma gondii first interacts with its host at the small intestinal epithelium. This interaction is crucial to controlling initial invasion and replication, as well as shaping the quality of the systemic immune response. It is therefore an attractive target for the design of novel vaccines and adjuvants. However, due to a lack of tractable infection models, we understand surprisingly little about the molecular pathways that govern this interaction. The in vitro culture of small intestinal epithelium as 3D enteroids shows great promise for modeling the epithelial response to infection. However, the enclosed luminal space makes the application of infectious agents to the apical epithelial surface challenging. Here, we have developed three novel enteroid-based techniques for modeling T. gondii infection. In particular, we have adapted enteroid culture protocols to generate collagen-supported epithelial sheets with an exposed apical surface. These cultures retain epithelial polarization, and the presence of fully differentiated epithelial cell populations. They are susceptible to infection with, and support replication of, T. gondii. Using quantitative label-free mass spectrometry, we show that T. gondii infection of the enteroid epithelium is associated with up-regulation of proteins associated with cholesterol metabolism, extracellular exosomes, intermicrovillar adhesion, and cell junctions. Inhibition of host cholesterol and isoprenoid biosynthesis with Atorvastatin resulted in a reduction in parasite load only at higher doses, indicating that de novo synthesis may support, but is not required for, parasite replication. These novel models therefore offer tractable tools for investigating how interactions between T. gondii and the host intestinal epithelium influence the course of infection