A Rapid Human Lung Tissue Dissociation Protocol Maximizing Cell Yield and Minimizing Cellular Stress.

The human lung is a complex organ comprised of diverse populations of epithelial, mesenchymal, vascular and immune cells, which gains even greater complexity during disease states. To effectively study the lung at a single cell level, a dissociation protocol that achieves the highest yield of viable cells of interest with minimal dissociation-associated protein or transcription changes key. Here, we detail a rapid collagenase-based dissociation protocol (Col-Short), which provides a high-yield single cell suspension suitable for a variety of downstream applications. Diseased human lung explants were obtained and dissociated through the Col-Short protocol and compared to four other dissociation protocols. Resulting single cell suspensions were then assessed with flow cytometry, differential staining, and quantitative real-time PCR to identify major hematopoietic and non-hematopoietic cell populations, as well as their activation states. We observed that the Col-Short protocol provides the greatest number of cells per gram of lung tissue with no reduction in viability when compared to previously described dissociation protocols. Col-Short had no observable surface protein marker cleavage as well as lower expression of protein activation markers and stress-related transcripts compared to four other protocols. The Col-Short dissociation protocol can be used as a rapid strategy to generate single cells for respiratory cell biology research.

Opioid initiation timing and palliative care referrals in advanced cancer: retrospective study.

OBJECTIVES: Early opioid initiation is recommended for advanced cancer pain, however the timing of opioid commencement in relation to diagnosis has not been described, and the role of palliative care prescribers is unclear. This study aims to determine the timing of opioid initiation by prescriber and cancer type in relation to key timepoints in the cancer illness course (diagnosis, palliative care referral and death). METHODS: This retrospective cohort study included patients at a quaternary cancer centre with incurable advanced cancer of five different subtype groups. Demographics, clinical characteristics, health service use and details of first slow and immediate release opioid prescription are reported. RESULTS: Among 200 patients, median time to first immediate release opioid prescription was 23 days (IQR 1-82) and to slow release opioid prescription was 47 days (IQR 14-155). Most patients (95%, (n=190) were referred to palliative care (median time to referral 54 days (IQR 18-190)). Non-palliative care prescribers initiated slow release opioids for half the cohort (49%, n=97) prior to referral. Patients with pancreatic cancer had the shortest time to slow/immediate release opioid prescription (median 10 days (IQR 0-39) and 26 days (IQR 1-43) respectively) and shortest survival (median 136 days (IQR 82-214)). CONCLUSIONS: Median time to opioid commencement was approximately 3 weeks after diagnosis. Despite early palliative care involvement, opioid initiation by non-palliative care clinicians was common and remains important. Timely palliative care referral for those with pancreatic cancer may include consideration of earlier complex pain presentations and shorter prognosis.

Diffusion Tensor MRI is sensitive to fibrotic injury in a mouse model of oxalate induced chronic kidney disease.

Chronic kidney disease (CKD) is characterized by inflammation and fibrosis in the kidney. Renal biopsies and estimated glomerular filtration rate (eGFR) remain the standard of care, but these endpoints have limitations in detecting the stage, progression, and spatial distribution of fibrotic pathology in the kidney. MRI diffusion tensor imaging (DTI) has emerged as a promising non-invasive technology to evaluate renal fibrosis in vivo both in clinical and preclinical studies. However, these imaging studies have not systematically identified fibrosis particularly deeper in the kidney where biopsy sampling is limited, or completed an extensive analysis of whole organ histology, blood biomarkers, and gene expression to evaluate the relative strengths and weaknesses of MRI for evaluating renal fibrosis. In this study, we performed DTI in the sodium oxalate mouse model of CKD. The DTI parameters fractional anisotropy, apparent diffusion coefficient, and axial diffusivity were compared between the control and oxalate groups with region-of-interest (ROI) analysis to determine changes in the cortex and medulla. Additionally, voxel-based analysis (VBA) was implemented to systematically identify local regions of injury over the whole kidney. DTI parameters were found to be significantly different in the medulla by both ROI analysis and VBA, which also spatially matched with collagen III IHC. The DTI parameters in this medullary region exhibited moderate to strong correlations with histology, blood biomarkers, hydroxyproline and gene expression. Our results thus highlight the sensitivity of DTI to the heterogeneity of renal fibrosis and importance of whole kidney non-invasive imaging.

Effect of Cangrelor on Infarct Size in ST-Segment-Elevation Myocardial Infarction Treated by Primary Percutaneous Coronary Intervention: A Randomized Controlled Trial (The PITRI Trial).

BACKGROUND: The administration of intravenous cangrelor at reperfusion achieves faster onset of platelet P2Y12 inhibition than oral ticagrelor and has been shown to reduce myocardial infarction (MI) size in the preclinical setting. We hypothesized that the administration of cangrelor at reperfusion will reduce MI size and prevent microvascular obstruction in patients with ST-segment-elevation MI undergoing primary percutaneous coronary intervention. METHODS: This was a phase 2, multicenter, randomized, double-blind, placebo-controlled clinical trial conducted between November 2017 to November 2021 in 6 cardiac centers in Singapore. Patients were randomized to receive either cangrelor or placebo initiated before the primary percutaneous coronary intervention procedure on top of oral ticagrelor. The key exclusion criteria included presenting <6 hours of symptom onset; previous MI and stroke or transient ischemic attack; on concomitant oral anticoagulants; and a contraindication for cardiovascular magnetic resonance. The primary efficacy end point was acute MI size by cardiovascular magnetic resonance within the first week expressed as percentage of the left ventricle mass (%LVmass). Microvascular obstruction was identified as areas of dark core of hypoenhancement within areas of late gadolinium enhancement. The primary safety end point was Bleeding Academic Research Consortium-defined major bleeding in the first 48 hours. Continuous variables were compared by Mann-Whitney U test (reported as median [first quartile-third quartile]), and categorical variables were compared by Fisher exact test. A 2-sided P<0.05 was considered statistically significant. RESULTS: Of 209 recruited patients, 164 patients (78%) completed the acute cardiovascular magnetic resonance scan. There were no significant differences in acute MI size (placebo, 14.9% [7.3-22.6] %LVmass versus cangrelor, 16.3 [9.9-24.4] %LVmass; P=0.40) or the incidence (placebo, 48% versus cangrelor, 47%; P=0.99) and extent of microvascular obstruction (placebo, 1.63 [0.60-4.65] %LVmass versus cangrelor, 1.18 [0.53-3.37] %LVmass; P=0.46) between placebo and cangrelor despite a 2-fold decrease in platelet reactivity with cangrelor. There were no Bleeding Academic Research Consortium-defined major bleeding events in either group in the first 48 hours. CONCLUSIONS: Cangrelor administered at the time of primary percutaneous coronary intervention did not reduce acute MI size or prevent microvascular obstruction in patients with ST-segment-elevation MI given oral ticagrelor despite a significant reduction of platelet reactivity during the percutaneous coronary intervention procedure. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03102723.

Intravenous iron for heart failure, iron deficiency definitions, and clinical response: the IRONMAN trial.

BACKGROUND AND AIMS: What is the relationship between blood tests for iron deficiency, including anaemia, and the response to intravenous iron in patients with heart failure? METHODS: In the IRONMAN trial, 1137 patients with heart failure, ejection fraction ≤ 45%, and either serum ferritin < 100 µg/L or transferrin saturation (TSAT) < 20% were randomized to intravenous ferric derisomaltose (FDI) or usual care. Relationships were investigated between baseline anaemia severity, ferritin and TSAT, to changes in haemoglobin from baseline to 4 months, Minnesota Living with Heart Failure (MLwHF) score and 6-minute walk distance achieved at 4 months, and clinical events, including heart failure hospitalization (recurrent) or cardiovascular death. RESULTS: The rise in haemoglobin after administering FDI, adjusted for usual care, was greater for lower baseline TSAT (Pinteraction < .0001) and ferritin (Pinteraction = .028) and more severe anaemia (Pinteraction = .014). MLwHF scores at 4 months were somewhat lower (better) with FDI for more anaemic patients (overall Pinteraction = .14; physical Pinteraction = .085; emotional Pinteraction = .043) but were not related to baseline TSAT or ferritin. Blood tests did not predict difference in achieved walking distance for those randomized to FDI compared to control. The absence of anaemia or a TSAT ≥ 20% was associated with lower event rates and little evidence of benefit from FDI. More severe anaemia or TSAT < 20%, especially when ferritin was ≥100 µg/L, was associated with higher event rates and greater absolute reductions in events with FDI, albeit not statistically significant. CONCLUSIONS: This hypothesis-generating analysis suggests that anaemia or TSAT < 20% with ferritin > 100 µg/L might identify patients with heart failure who obtain greater benefit from intravenous iron. This interpretation requires confirmation.

Thyroid hormone protects human lung epithelial cells from cold preservation and warm reperfusion-induced injury.

BACKGROUND: Cellular stress associated with static-cold storage (SCS) and warm reperfusion of donor lungs can contribute to ischemia-reperfusion (IR) injury during transplantation. Adding cytoprotective agents to the preservation solution may be conducive to reducing graft deterioration and improving post-transplant outcomes. METHODS: SCS and warm reperfusion were simulated in human lung epithelial cells (BEAS-2B) by exposing cells to low potassium dextran glucose solution at 4 °C for different periods and then switching back to serum-containing culture medium at 37 °C. Transcriptomic analysis was used to explore potential cytoprotective agents. Based on its results, cell viability, caspase activity, cell morphology, mitochondrial function, and inflammatory gene expression were examined under simulated IR conditions with or without thyroid hormones (THs). RESULTS: After 18 h SCS followed by 2 h warm reperfusion, genes related to inflammation and cell death were upregulated, and genes related to protein synthesis and metabolism were downregulated in BEAS-2B cells, which closely mirrored gene profiles found in thyroid glands of mice with congenital hypothyroidism. The addition of THs (T3 or T4) to the preservation solution increases cell viability, inhibits activation of caspase 3, 8 and 9, preserves cell morphology, enhances mitochondrial membrane potential, reduces mitochondrial superoxide production, and suppresses inflammatory gene expression. CONCLUSION: Adding THs to lung preservation solutions may protect lung cells during SCS by promoting mitochondrial function, reducing apoptosis, and inhibiting pro-inflammatory pathways. Further in vivo testing is warranted to determine the potential clinical application of adding THs as therapeutics in lung preservation solutions.

Partially Ablative Body Radiotherapy (PABR): A novel approach for palliative radiotherapy of locally advanced bulky unresectable sarcomas.

BACKGROUND: Locally advanced, bulky, unresectable sarcomas cause significant tumour mass effects, leading to burdensome symptoms. We have developed a novel Partially Ablative Body Radiotherapy (PABR) technique that delivers a high, ablative dose to the tumour core and a low, palliative dose to its periphery aiming to increase overall tumour response without significantly increasing treatment toxicity. AIM: This study aims to report the safety and oncologic outcomes of PABR in patients with bulky, unresectable sarcomas. METHODS AND MATERIALS: A total of 18 patients with histologically proven sarcoma treated with PABR from January 2020 to October 2023 were retrospectively reviewed. The primary endpoints were symptomatic and structural response rates. Secondary endpoints were overall survival, freedom from local progression, freedom from distant progression, and acute and late toxicity rates. RESULTS: All patients had tumours ≥5 cm with a median tumour volume of 985 cc, and the most common symptom was pain. The median age is 72.5 years and 44.5 % were ECOG 2-3. The most common regimen used was 20 Gy in 5 fractions with an intratumoral boost dose of 50 Gy (83.3 %). After a median follow-up of 11 months, 88.9 % of patients exhibited a partial response with a mean absolute tumour volume reduction of 49.5 %. All symptomatic patients experienced symptom improvement. One-year OS, FFLP and FFDP were 61 %, 83.3 % and 34.8 %, respectively. There were no grade 3 or higher toxicities. CONCLUSION: PABR for bulky, unresectable sarcomas appears to be safe and may provide good symptomatic response, tumour debulking, and local control. Further study is underway.

Opioid Switch Dosing in Chronic Cancer Pain: A Prospective Longitudinal Study.

Background: Opioid switching is common, however, conversion tables have limitations. Guidelines suggest postswitch dose reduction, yet, observations show opioid doses may increase postswitch. Objectives: To document the opioid conversion factor postswitch in cancer, and whether pain and adverse effect outcomes differ between switched opioid groups. Design/Setting: This multicenter prospective longitudinal study included people with advanced cancer in Australia. Clinical data (demographics, opioids) and validated instruments (pain, adverse effects) were collected twice, seven days apart. Results: Opioid switch resulted in dose increase (median oral morphine equivalent daily dose 90 mg [interquartile range {IQR} 45-184] to 150 mg [IQR 79-270]), reduced average pain (5.1 [standard deviation {SD} 1.7] to 3.8 [SD 1.6]), and reduced adverse effects. Hydromorphone dose increased 2.5 times (IQR 1.0-3.6) above the original conversion factor used. Conclusions: Opioid switching resulted in overall dose increase, particularly when switching to hydromorphone. Higher preswitch dosing may require higher dose conversion ratios. Dose reduction postswitch risks undertreatment and may not be always appropriate.

Headplate Installation and Craniotomy for Awake In Vivo Electrophysiological Recordings or Two-Photon Imaging of the Mouse Inferior Colliculus.

The inferior colliculus (IC) is an important processing center in the auditory system, which also receives non-auditory sensory input. The IC consists of several subnuclei whose functional role in (non-) auditory processing and plastic response properties are best approached by studying awake animals, preferably in a longitudinal fashion. The increasing use of mice in auditory research, the availability of genetic models, and the superficial location of the IC in the mouse have made it an attractive species for studying IC function. Here, we describe a protocol for exposing the mouse IC for up to a few weeks for in vivo imaging or electrophysiology in a stable manner. This method allows for a broader sampling of the IC while maintaining the brain surface in good quality and without reopening the craniotomy. Moreover, as it is adaptable for both electrophysiological recordings of the entire IC and imaging of the dorsal IC surface, it can be applied to answer a multitude of questions. Key features • A surgical protocol for long-term physiological recordings from the same or separate neuronal populations in the inferior colliculus. • Optimized for awake in vivo experiments in the house mouse (Mus musculus).

Effect of gene variants on opioid dose, pain and adverse effect outcomes in advanced cancer: an explorative study.

Aim: Associations between gene variants and opioid net effect are unclear. We conducted an exploratory pharmacogenetic analysis of 35 gene variants and opioid response in advanced cancer. Patients & methods: This multi-center prospective cohort study included clinical data, questionnaires (pain and adverse effects) and DNA (blood). Negative binomial regression and logistic regression were used. Results: Within 54 participants, eight statistically significant associations (p = 0.002-0.038) were observed between gene variants and opioid dose, pain scores or adverse effects, the majority being within the neuroimmune TLR4 pathway (IL1B [rs1143634], IL2 [rs2069762], IL6 [rs1800795], BDNF [rs6265]) and ARRB2 pathway (ARRB2 [rs3786047], DRD2 [rs6275]). Conclusion: Neuroimmune pathway genes may contribute to differences in opioid response in cancer and may be included in future similar studies.

Acellular ex vivo lung perfusate silences pro-inflammatory signaling in human lung endothelial and epithelial cells.

BACKGROUND: Ischemia-reperfusion injury is a key complication following lung transplantation. The clinical application of ex vivo lung perfusion (EVLP) to assess donor lung function has significantly increased the utilization of "marginal" donor lungs with good clinical outcomes. The potential of EVLP on improving organ quality and ameliorating ischemia-reperfusion injury has been suggested. METHODS: To determine the effects of ischemia-reperfusion and EVLP on gene expression in human pulmonary microvascular endothelial cells and epithelial cells, cell culture models were used to simulate cold ischemia (4 °C for 18 h) followed by either warm reperfusion (DMEM + 10% FBS) or EVLP (acellular Steen solution) at 37 °C for 4 h. RNA samples were extracted for bulk RNA sequencing, and data were analyzed for significant differentially expressed genes and pathways. RESULTS: Endothelial and epithelial cells showed significant changes in gene expressions after ischemia-reperfusion or EVLP. Ischemia-reperfusion models of both cell types showed upregulated pro-inflammatory and downregulated cell metabolism pathways. EVLP models, on the other hand, exhibited downregulation of cell metabolism, without any inflammatory signals. CONCLUSION: The commonly used acellular EVLP perfusate, Steen solution, silenced the activation of pro-inflammatory signaling in both human lung endothelial and epithelial cells, potentially through the lack of serum components. This finding could establish the basic groundwork of studying the benefits of EVLP perfusate as seen from current clinical practice.

Defining research priorities and needs in cancer symptoms for adults diagnosed with cancer: an Australian/New Zealand modified Delphi study.

PURPOSE: This study asked consumers (patients, carers) and healthcare professionals (HCPs) to identify the most important symptoms for adults with cancer and potential treatment interventions. METHODS: A modified Delphi study was conducted involving two rounds of electronic surveys based on prevalent cancer symptoms identified from the literature. Round 1 gathered information on participant demographics, opinions and/or experience on cancer symptom frequency and impact, and suggestions for interventions and/or service delivery models for further research to improve management of cancer symptoms. In Round 2, respondents ranked the importance of the top ten interventions identified in Round 1. In Round 3, separate expert panels of consumers and healthcare professionals (HCPs) attempted to reach consensus on the symptoms and interventions previously identified. RESULTS: Consensus was reached for six symptoms across both groups: fatigue, constipation, diarrhoea, incontinence, and difficulty with urination. Notably, fatigue was the only symptom to reach consensus across both groups in Round 1. Similarly, consensus was reached for six interventions across both groups. These were the following: medicinal cannabis, physical activity, psychological therapies, non-opioid interventions for pain, opioids for breathlessness and cough, and other pharmacological interventions. CONCLUSIONS: Consumers and HCPs prioritise differently; however, the symptoms and interventions that reached consensus provide a basis for future research. Fatigue should be considered a high priority given its prevalence and its influence on other symptoms. The lack of consumer consensus indicates the uniqueness of their experience and the need for a patient-centred approach. Understanding individual consumer experience is important when planning research into better symptom management.

Effectiveness of Opioid Switching in Advanced Cancer Pain: A Prospective Observational Cohort Study.

Opioid switching is a common practice of substituting one opioid for another to improve analgesia or adverse effects; however, it has limited evidence. This study aimed to examine the effectiveness of opioid switching in advanced cancer. This multi-center prospective cohort study recruited patients assessed to switch opioids (opioid switch group) or to continue ongoing opioid treatment (control group). Clinical data (demographics, opioids) and validated instruments (pain and adverse effects) were collected over two timepoints seven days apart. Descriptive analyses were utilized. Non-parametric tests were used to determine differences. Fifty-four participants were recruited (23 control group, 31 switch group). At the follow-up, opioid switching reduced pain (worst, average, and now) (p < 0.05), uncontrolled breakthrough pain (3-fold reduction, p = 0.008), and psychological distress (48% to 16%, p < 0.005). The switch group had a ≥25% reduction in the reported frequency of seven moderate-to-severe adverse effects (score ≥ 4), compared to a reduction in only one adverse effect in the control group. The control group experienced no significant pain differences at the follow-up. Opioid switching is effective at reducing pain, adverse effects, and psychological distress in a population with advanced cancer pain, to levels of satisfactory symptom control in most patients within 1 week.

Prevalence, Severity, and Predictors of Insomnia in Advanced Colorectal Cancer.

CONTEXT: Insomnia is an under-recognized and undertreated symptom in palliative care and advanced cancer cohorts. Insomnia in an advanced colorectal cancer cohort is yet to be investigated despite colorectal cancer being the third commonest cancer worldwide and one with a high symptom burden. OBJECTIVES: To examine the prevalence of insomnia and its associations in a large advanced colorectal cancer cohort. METHODS: A consecutive cohort study of 18,302 patients with colorectal cancer seen by palliative care services across various settings (inpatient, outpatient, and ambulatory) was conducted from an Australia-wide database (2013-2019). The Symptom Assessment Score (SAS) was used to assess the severity of insomnia. Clinically significant insomnia was defined as SAS score ≥3/10, and used to compare associations with other symptoms and functional scores from validated questionnaires. RESULTS: The prevalence of any insomnia was 50.5%, and clinically significant insomnia 35.6%, particularly affecting people who were younger (<45-years-old), more mobile (AKPS score ≥70), or physically capable (RUG-ADL score ≤5). Outpatients and patients living at home had higher prevalence of insomnia. Nausea, anorexia and psychological distress were the commonest concurrent symptoms in patients with clinically significant insomnia. CONCLUSIONS: To our knowledge, this study was the first to investigate the prevalence and associations of insomnia in an advanced colorectal cancer cohort. Our findings demonstrate several groups at greater risk of suffering from insomnia (younger, greater physical capacity, living at home, and those with greater psychological distress). This may guide earlier recognition and management of insomnia to improve overall quality of life in this population.

LOXL4, but not LOXL2, is the critical determinant of pathological collagen cross-linking and fibrosis in the lung.

Idiopathic pulmonary fibrosis is a progressive fibrotic disease characterized by excessive deposition of (myo)fibroblast produced collagen fibrils in alveolar areas of the lung. Lysyl oxidases (LOXs) have been proposed to be the central enzymes that catalyze the cross-linking of collagen fibers. Here, we report that, while its expression is increased in fibrotic lungs, genetic ablation of LOXL2 only leads to a modest reduction of pathological collagen cross-linking but not fibrosis in the lung. On the other hand, loss of another LOX family member, LOXL4, markedly disrupts pathological collagen cross-linking and fibrosis in the lung. Furthermore, knockout of both Loxl2 and Loxl4 does not offer any additive antifibrotic effects when compared to Loxl4 deletion only, as LOXL4 deficiency decreases the expression of other LOX family members including Loxl2. On the basis of these results, we propose that LOXL4 is the main LOX activity underlying pathological collagen cross-linking and lung fibrosis.

Does cancer type and adjuvant analgesic prescribing influence opioid dose?-a retrospective cross-sectional study.

Opioids are the backbone of cancer pain management. Minimal evidence exists examining the relationship between cancer type and opioid dose. Similarly, the use of adjuvant analgesics and its impact within an inpatient cancer setting is understudied. This study examined the influence of cancer type upon opioid dose, measured by oral morphine equivalent daily dose (oMEDD). The effect of adjuvant analgesics on patient oMEDD was also examined. This retrospective cross-sectional study examined records of 520 patients admitted to Royal Melbourne Hospital or Peter MacCallum Cancer Centre between 2016 and 2018 with advanced cancer. Number and dose of both opioid and adjuvant analgesics were collected along with demographic and cancer data. Comparisons of median oMEDD by cancer type [analysis of variance (ANOVA), non-parametric t-tests] and adjuvant analgesics (Kruskal-Wallis test) were performed. There were no statistically significant differences in oMEDD between the 12 cancer types (P=0.83; n=215). Patients co-prescribed pregabalin (n=102) and paracetamol (n=73) as adjuvant analgesics were on significantly higher daily oMEDD [60 mg (P=0.015), 90 mg (P<0.001), respectively]. Opioid dose did not differ significantly between cancer types. The observed use of adjuvant analgesics coincided with significantly higher oMEDD prescription which may relate to complex pain seen in this cohort of inpatients in a quarternary cancer centre. Future research should focus on pain type and aetiology, and pain scores in different cancer pain syndromes to determine the net effect of opioids and adjuvants in cancer pain prescribing.

Establishing a Longitudinal Opioid Pharmacogenomic Registry for Cancer Patients: Feasibility and Acceptability.

Purpose: Individual genetic variation can affect both pain expression and opioid response. Large cohort datasets are required to validate evidence influencing genomic factors in opioid response. This study examined the feasibility of establishing an opioid pharmacogenomics registry for cancer patients containing longitudinal matched clinical, symptom, pharmacological, and genomic data, with an a priori feasibility target of 50 participants within 12 months. Methods: Consecutive patients with advanced cancer receiving opioids across five palliative care services were recruited. Clinical data (demographics, pain data, adverse effects, medications) and blood (DNA, RNA, pharmacokinetics) were collected over two time points. Patient and clinician qualitative interviews were conducted to assess acceptability. This study was approved by the SVHA Ethics Committee, Melbourne, Australia (HREC 252/18). Results: Enrollment for the registry was deemed feasible. Fifty-eight participants were recruited (median age 63.7, 45% female, 83% complete data), with the most frequent diagnosis being lung cancer (n = 18, 33%) and oxycodone the most frequently prescribed opioid (n = 30, 52%). Qualitative data indicated positive engagement from both patients and clinicians. Conclusion: Establishing a longitudinal opioid pharmacogenomic registry in patients with cancer receiving palliative care is feasible and readily acceptable.