Rationale: Cigarette smoking is a well-established risk factor for myocardial infarction and sudden cardiac death. The deleterious effects are mainly due to nicotine, but the mechanisms involved and theranostics remain unclear. Thus, we tested the hypothesis that nicotine exposure increases the heart sensitivity to ischemia/reperfusion injury and dysfunction, which can be rescued by autophagy inhibitor.Methods: Nicotine or saline was administered to adult rats via subcutaneous osmotic minipumps in the absence or presence of an autophagy inhibitor, 3-methyladenine (3-MA). After 30 days of nicotine treatment, the rats underwent the cardiac ischemia/reperfusion (I/R) procedure and echocardiography analysis, and the heart tissues were isolated for molecular biological studies.Results: Nicotine exposure increased I/R-induced cardiac injury and cardiac dysfunction as compared to the control. The levels of autophagy-related proteins including LC3 II, P62, Beclin1, and Atg5 were upregulated in the reperfused hearts isolated from nicotine-treated group. In addition, nicotine enhanced cardiac and plasma ROS production, and increased the phosphorylation of GSK3β (ser9) in the left ventricle tissues. Treatment with 3-MA abolished nicotine-mediated increase in the levels of autophagy-related proteins and phosphorylation of GSK3β, but had no effect on ROS production. Of importance, 3-MA ameliorated the augmented I/R-induced cardiac injury and dysfunction in the nicotine-treated group as compared to the control.Conclusion: Our results demonstrate that nicotine exposure enhances autophagy signaling pathway, resulting in development of ischemic-sensitive phenotype of heart. It suggests a potentially novel therapeutic strategy of autophagy inhibition for the treatment of ischemic heart disease. Epidemiologic studies have shown that cigarette smoking is the most prominent risk factor for coronary heart disease and cardiovascular mortality in the general population Nicotine exposure, either from tobacco cigarette smoking or nicotine use (nicotine gum or patch) has become one of the most pressing public concerns in modern life. Furthermore, electronic cigarette (e-cigarette), an electronic nicotine delivery system has been introduced in the global market during the past ten years. Thus, nicotine addiction and nicotine abuse are becoming a potential public health concern in the 21Autophagy is a cellular process associated with unnecessary protein damage and organelle degradation. Autophagy functions as a protein quality controller and cellular homeostasis keeper Therefore, in the present study we first evaluated whether chronic nicotine exposure increased the heart susceptibility to ischemia/reperfusion (I/R) injury and decreased heart functional recovery after I/R injury. Next, we examined whether chronic nicotine exposure enhanced autophagy-related biomarkers in cardiomyocytes. Finally, we assessed the effect of therapeutic inhibition of autophagy activation on nicotine-mediated cardiac ischemic injury and dysfunction to test the hypothesis that nicotine-mediated autophagy plays a causal role in the development of heart ischemic-sensitive phenotype in an adult rat model. Our findings suggest that autophagy may be a potential therapeutic molecular target for the treatment of cardiovascular ischemic disease.ad libitum. Rats were anesthetized with isoflurane  and adequate anesthesia was determined by the loss of pedal withdrawal reflex and other reactions from the animals in response to pinching the toe or tail. Saline or nicotine (at 4 µg/kg/min) were individually administered to rats through osmotic minipumps  and inserted under the skin on the back of rats for 30 days, as previously described in detail The procedures and protocols were approved by the Institutional Animal Care and Use Committee of Loma Linda University. All animal studies followed the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Sprague-Dawley male rats (8-months-old) were purchased from Charles River Laboratories  and housed individually in Plexiglas acrylic cages, located in air-conditioned rooms . Pellet food and tap water were available To measure LV function and heart chamber dimensions in the four groups of animals, echocardiography was performed with LOGIQ e Ultrasound  as previously described in vivo as described previously After 30 days of nicotine treatment, the rats were subjected to ischemia/reperfusion (I/R) procedure Masson's trichrome staining is widely used to study cardiac pathologies including cardiac infarction and fibrosis. In the present study, the heart was rapidly excised, rinsed to remove blood. Then, the heart tissues were cryopreserved by using optimal cutting temperature (OCT) tissue medium and sectioned transversely from the basal part to the apex of left ventricle using a cryostat with 10 µm thickness. Masson's Trichrome staining (Abcam) was performed according to the manufacture's instruction to quantify infarct scar size (blue staining). The infarct scar size was analyzed by computerized planimetry and expressed as the ratio of scar area to total LV area.in situ, as described previously The oxidative fluorescent dye hydroethidium (HE) was used to evaluate superoxide production TMin vitro ROS/RNS assay kit , following the manufacturer's instruction and described previously Plasma was collected from the rat after seven days of reperfusion. The total ROS levels in the plasma were measured with the OxiselectProtein abundance in heart was measured as previously described All data are expressed as the mean ± SEM obtained from the number (n) of experimental animals given. Differences between the groups were compared by Student's t-test or analysis of variance (ANOVA) using the Graph-Pad Prism software , where appropriate. For all comparisons, P-values less than 0.05 indicated statistical significance.Chronic nicotine treatment for 30 days had no effect on the body weights of rats (data not shown). Similarly, there was no significant difference in the ratio of heart to body weight between the saline control and nicotine-treated groups (data not shown). As shown in Fig. Fig. Chronic nicotine exposure significantly enhanced the LC3 II/LC3 I protein expression ratio Fig. A and p622-) was assessed by the oxidative fluorescent dye HE. As shown in Fig. 2- in the LV tissues as compared with the saline control groups in the absence of 3-MA under I/R procedure. Interestingly, 3-MA treatment did not affect nicotine-mediated increase in O2- production under I/R procedure. In addition, we determined the ROS production in the plasma using a ROS assay kit under I/R procedure. As shown in Fig. To determine whether chronic nicotine exposure causes oxidative damage in the heart, production of superoxide anion  inhibition of autophagy with an autophagy inhibitor normalized the aberrant autophagy level and reversed the increased vulnerability to heart I/R injury and dysfunction. These findings suggest a potentially novel therapeutic strategy of anti-autophagy for the treatment of smoking/nicotine-mediated heart ischemic disease.Epidemiological and animal studies have shown that tobacco smoking increases the risk of ischemic heart disease Our data indicated that the ratio of heart weight to body weight was not changed between the control and nicotine-treated groups. In addition, although we did not measure the ratio of heart weight/body weight after seven days of reperfusion, our echocardiographic observations indicated that there were no differences of LVPWd (data not shown) between the nicotine-treated and control groups. These data suggest that nicotine does not induce cardiac hypertrophy at both resting and I/R-challenged condition. The present study showed that chronic nicotine exposure had no significant effect on pre-ischemic baseline values of heart function determined by echocardiography but significantly increased LV myocardial infarct size and impaired LV function after ischemia/reperfusion in rats. The data suggests that chronic nicotine exposure does not affect heart function at normal physiologic conditions but alters the heart function when it encounters an ischemic stress challenge. Previous studies in human and animal models have reported that cigarette smoking is one of the top risk factors of ischemic heart disease in vitro have demonstrated that treatment of 3-MA or knockdown of Beclin 1 inhibits ischemia/ reperfusion-induced autophagy, leading to reduced cardiac injury and enhanced cardiac myocyte survival in vivo treatment of 3-MA had no effect on I/R-induced heart injury in saline control animals. This discrepant effect of 3-MA between in vitro cardiac myocytes and in vivo heart may suggest that basal autophagy dose not play a key role in I/R-induced heart injury in animals at physiologic condition. However, our present findings that that treatment with 3-MA significantly inhibited I/R-induced heart infarct size in nicotine-treated animals and eliminated the difference of I/R-induced cardiac infarct size and dysfunction between the saline control and nicotine-treated rats, suggest that the excess of autophagy plays an important role in nicotine-mediated I/R-induced heart ischemic injury and dysfunction. Taken together, our current findings with others suggest a causal effect of autophagy in the setting of ischemic heart injury and dysfunction under pathologic condition. In addition, we need keep in mind that autophagy signaling pathway may be not the only signaling pathway involved nicotine-mediated cardiac dysfunction, other signaling pathways such as apoptosis may also be involved.The molecular mechanisms underlying nicotine-mediated increase in ischemic injury and heart dysfunction are still largely unknown. In the present study, we observed that chronic nicotine exposure enhanced autophagy biomarkers  protein abundance in cardiac tissues. Consistent with our observation, other studies have shown that nicotine exposure increases autophagy activation in human fibroblast cells The process of autophagy is complex and mainly comprised of the following stages: initiation, nucleation, expansion, maturation, and degradation. Numerous autophagy-related (Atg) genes and signaling molecules have been identified and are sequentially activated to regulate different stages of autophagy. There are three classes of PtdIns3K and phosphatidylinositol 3-Kinases (PI3Ks) involved in the regulation of autophagy. Class I PI3K responds to growth factor signaling and produces PtdInsP3 that negatively regulates autophagy via the AKT-mTOR complex, whereas the class III PI3K, as well as class II PI3K, contributes to the initiation and progression of autophagy. Beclin 1 is one of the major genes involved in the class III PI3K complex signaling It has been shown that cigarette smoking increases the sensitivity of hearts to ischemia/ reperfusion injury associated with increased production of oxygen free radicals GSK3β gene plays a significant role in embryonic cardiomyocyte development. Recent studies demonstrate a functional role of GSK3β in negative modulation of cardiac hypertrophy and in the setting of cardiac ischemic disease In summary, the present study provides new evidence that excess autophagy is one of the key molecular signaling linkers between nicotine exposure and the development of the heart ischemic-sensitive phenotype. Our data indicated that nicotine exposure stimulated oxidative stress and enhanced cardiac autophagy biomarkers, resulting in increased ischemic heart infarct size and heart dysfunction. Of importance, inhibition of autophagy via 3-MA rescued nicotine-mediated pathological effects and heart dysfunctions. These findings not only provide novel evidence regarding the molecular mechanisms underlying nicotine-induced ischemic heart injury and dysfunction, but also suggest a potential therapeutic molecular target of autophagy involved in the setting of ischemic heart disease.In our present study we have demonstrated that chronic nicotine exposure enhances I/R-induced heart dysfunction associated with an excessive autophagy flux. However, one of the limitations for this study is unknown whether the induction of the autophagy proteins is directly regulated by nicotine exposure or I/R procedure. Therefore, in our future study we should measure the basal levels of autophagy influx in the control and nicotine-treated animals to see whether nicotine-mediated pre-existed increase of autophagy in the heart would exacerbate the ischemic/reperfusion injury. While infarct size and cardiac function are good indicators of myocardial ischemia/reperfusion injury, other indicators such as Troponin I concentration can further support the claim. Thus, in our future studies, we will investigate whether nicotine exposure affects Troponin I expression in response to I/R. It's well known that 3-MA is a selective autophagy/PI3K inhibitor. However, 3-MA could induce potential non-specific effects on heart function. It depends on the patho-physiologic condition of the animals and dosage of 3-MA. For instance, previous studies have reported that 3-MA has dual role in modulation of autophagy and induces an autophagic promotion effect under nutrient-rich condition


Growing evidence shows that individuals who have high levels of psychological resilience maintain higher levels of physical and psychological health in later life. Individuals cultivate psychological resilience over the life course, yet little research has explored its mechanistic effects on health during midlife. One source of resilience may be formal education, which is a well-established determinant of health in adulthood. Resilience might be one reason for this robust association, as education helps individuals develop greater psychological resources in adulthood. On the other hand, having a college degree also increases access to other health-promoting resources that can be leveraged over the life course, such as better-paying and higher-quality jobs. Using data drawn from the National Longitudinal Study of Adolescent to Adult Health , the current paper examines: 1) how early-life factors shape psychological resilience in early adulthood (24-32 years); and 2) the effects of early adulthood resilience on the association between education and health in mid-life (36-44 years). Results show that psychological resilience and college education have significant direct effects on health in midlife, net of health in early adulthood. However, first-generation college graduates cultivate more psychological resilience from their educational attainment than do those with college-educated parents. That is, higher education serves as a leveler for health gaps in midlife for those with fewer resources available in early life by bolstering resilience. These results provide important insights about how early life factors play an important role in shaping successful aging processes.


Novel partial volume correction (PVC) algorithms have been validated by assuming ideal conditions of image processing; however, in real clinical PET studies, the input datasets include error sources which cause error propagation to the corrected outcome.18F]THK-5351 and to discuss the reliability of those algorithms for clinical applications. In order to mimic brain PET imaging of [18F]THK-5351, pseudo-observed SUVR images for one healthy adult and one adult with Alzheimer’s disease were simulated from individual PET and MR images. The partial volume effect of pseudo-observed PET images were corrected by using Müller-Gärtner (MG), the geometric transfer matrix (GTM), Labbé (LABBE), regional voxel-based (RBV), iterative Yang (IY), structural functional synergy for resolution recovery (SFS-RR), and modified SFS-RR algorithms with incorporation of error sources in the datasets for PVC processing. Assumed error sources were mismatched FWHM, inaccurate image-registration, and incorrectly segmented anatomical volume. The degree of error propagations in ROI values was evaluated by percent differences (%diff) of PV-corrected SUVR against true SUVR.We aimed to evaluate error propagations of seven PVCs algorithms for brain PET imaging with THK-5351 were significant. Robust algorithms for clinical applications must be carefully selected according to the study design of clinical PET data.Error propagations of 7 PVC algorithms for brain PET imaging with [ Positron emission computed tomography (PET) enables quantification of the biodistribution of administrated radiopharmaceuticals in vivo. However, the PET images suffer from the partial volume effect (PVE) due to the limited spatial resolution of PET scanners, where regional depiction of the uptake of PET radiopharmaceutical is blurred and its quantitative accuracy is reduced. Correction of PVE, known as partial volume correction (PVC), is usually performed for PET images by using spread function of blurring and an anatomical prior during post-reconstruction processing or during image reconstruction .Post-reconstruction PVC, together with anatomical information based on magnetic resonance (MR) image, has been distributed as commercial or academic software and used for clinical studies due to easy realization and recent advances of hybrid PET-MRI scanners , 3. In gFor the diagnosis of early Alzheimer’s disease (AD), the in vivo PET imaging of characteristic pathological features aggregated amyloid protein (Aβ) and neurofibrillary tangle (tau) by using dedicated radiopharmaceuticals has been recognized as potentially having an important role . HoweverThere have been several reports which stated that application of PVC for of tau and amyloid PET imaging improved the accuracy and precision of the quantification of radiopharmaceutical-uptake –18. Lope18F]FDG images by eight PVC algorithms [18F]FDOPA images, PV correction by GTM algorithm together with inaccurate image registration between PET and MRI reduced the quantitative accuracy of radioactivity concentration in the region of interests (ROI) [18F]THK-5351 imaging.There have been several reports about error propagation of PVC for brain PET imaging. It was reported that the incorrect specification of the full-width of half-maximum (FWHM) parameter with wider or narrower spread function of blurring relative to the true spread function resulted in the systematic over or underestimation in the correction of simulated [gorithms . For simts (ROI) . Further18F]THK-5351 in our previous study THK-5351 SUVR images of each one healthy, elderly individual and one elderly individual with typical AD were numerically generated . In our processing, scatter coincidences (estimated by single scatter simulation) and random coincidences (achieved by constant random background) were once added into the forward projected sinogram and subtracted from the noisy sinogram. The noise level of the image reconstructed from the noisy sinogram was finally adjusted with scaled Poisson noise to achieve a similar noise level to that of clinical PET images. The attenuation map of 511 keV gamma ray  was created by segmenting T1-weighted MR images with a pseudo-CT synthesis tool (http://niftyweb.cs.ucl.ac.uk/), and then the segmented images were carefully checked and manually re-drawn by an experienced physician  and was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all subjects after a complete description of the study had been provided.Seven partial volume correction algorithms were implemented for pseudo-observed PET SUVR images. The detail of each algorithm is described in , 25 and https://github.com/UCL/PETPVC) THK-5351 were normalized with the ROI value of the cerebellar grey matter. For error propagation analysis, 6 ROIs  were selected  Raclopride THK-5351 under the FBP reconstruction algorithm, and the outcome measure was SUVR. So, the %differences, which we reported in this study, may differ with other anatomies and the SUVR distribution/value itself. However, we believe that the present error propagation study of [18F]THK-5351 will contribute to both the understanding and prevention of the error propagation phenomena in future clinical PET data analysis. Another limitation is that we only evaluated the unique error sources and did not evaluate multiple error sources in combination. Possible combination may be segmentation error and registration error. Our results suggested that if both regional segmentation error and systematic registration error are simultaneously present in input data sets for PVC processing, most regions except for the few that have been incorrectly segmented will be affected only by registration error. Furthermore, we did not investigate the effect of intrinsic errors in the PET image itself, such as due to inaccurate attenuation correction. If the PET image includes systematic or random errors, these will be propagated to the outcome of the PVC.In this study, we simulated only one HC condition and one AD condition of THK-5351 were significant. The robust algorithms for clinical applications must be carefully selected according to the study design of clinical PET data.We demonstrated error propagation by numerical simulation of THK-PET imaging. The error propagations of seven PVCs algorithms for brain PET imaging with [


Correction to: BMC Health Serv Res 20, 589 (2020)https://doi.org/10.1186/s12913-020-05267-6Following publication of the original article , an errobold typeface.The updated paragraphs are given below and the changes have been highlighted in DesignIdentifying the current state of HSSR within the health systemDevelopment of the CHSSR-S strategyIntegration of the HSSR strategy within the health systemDevelopment of the Children’s Health Service and System Research Strategy (CHSSR-S) was informed by an inductive, bottom-up, participatory systems approach. This conceptual approach intended to place clinical, research and system leadership at the forefront of designing and implementing the CHSSR-S . This study was conducted across three key phases that were underpinned by our conceptual approach :Decision-making – what is the best care to provide?Implementation – how can we best provide this care?Evaluation – what are the outcomes resulting from this care?The overall structure and content of the CHSSR-S was aligned with three interrelated dimensions of care relevant to HSSR [10]:The original article  has been


Multiple combinational regimens have recently been approved and are now considered the standard of care for patients with advanced clear cell renal cell carcinoma (RCC). Several additional combinational regimens are deep in clinical assessment and are likely to soon join the crowded front-line therapeutic landscape. Most of these regimens are combinations of agents already approved as single-agents in RCC including tyrosine kinase inhibitors (TKI) and immune checkpoint inhibitors. While these new front-line regimens are associated with reliably high response rates and prolonged survival, complete and durable remissions remain limited to a small subset of patients and the vast majority of patients continue to require subsequent therapy. The need for the continued development of novel agents in RCC persists and efforts have focused on agents targeting the molecular biology of clear cell RCC and novel immunotherapies including cytokines. In this review, we discuss the progress in the development of these novel therapies in the context of the evolving standard of care for patients with advanced clear cell RCC. Renal cell carcinoma (RCC) is the seventh most common malignancy in the USA with over 73,000 cases and 14,830 deaths expected in 2020  –6. The vVHL gene, has been found in 70–90% of clear cell RCC and is believed to be the earliest event in tumorigenesis  in clear cell RCC not possessing alterations in VHL, suggesting that loss of function of the VHL complex is a central feature of nearly all clear cell RCC . These agents are outlined in The combination of nivolumab  targeting programmed death (PD)-1) and ipilimumab ) was assessed in a randomized Phase III trial versus sunitinib in patients with untreated advanced RCC . The iniThe combination of axitinib and pembrolizumab  was assessed in a randomized Phase III trial versus sunitinib in patients with advanced RCC KEYNOTE-426) ( (26). OvThe combination of axitinib and avelumab  was similarly assessed in a large randomized Phase III trial versus sunitinib in patients with advanced RCC in which 886 treatment-naïve patients were randomized to receive either the combination or sunitinib (JAVELIN). The study was designed with the primary endpoints of PFS and OS among patients with PD-1 Ligand (PD-L1) positive tumors. In the initial report of the results, the combination of axitinib and avelumab resulted in superior PFS in both patients with PD-L1 positive tumors  and the overall population  compared with sunitinib . The comThe most current standard care for the majority of patients with advanced clear cell RCC will be to start treatment with combinations of ipilimumab–nivolumab, axitinib–pembrolizumab, or axitinib–avelumab. However, as noted above, complete responses that are maintained off therapy are rare and the vast majority of patients will require subsequent lines of therapy. With the changes to frontline therapy being so recent, the most appropriate choice of second-line therapy remains unclear. A small retrospective analysis has shown that the combination of ipilimumab–nivolumab has moderate activity in patients treated with prior therapy including the combination of TKI–checkpoint inhibitors . As patiDetermination of the most appropriate sequence of the agents will require continued efforts to develop predictive biomarkers of response to current therapies. While significant effort has gone into identifying mechanisms of resistance to VEGF-targeted therapies, whether these same pathways will be relevant when VEGF-targeted therapies are combined with immune therapy remains unclear . SimilarSeveral important clinical trials are underway which may also contribute to the first-line RCC treatment paradigm . Many ofWhile the studies discussed thus far involve combinational regimens involving agents that are already approved in RCC, there are also numerous agents in clinical development in clear cell RCC with completely novel mechanisms of action. Like the VEGF-targeted therapies, many of these novel agents target therapeutic vulnerabilities engendered by the molecular biology of clear cell RCC.The novel agents perhaps most centrally targeted to the molecular biology of clear cell RCC are inhibitors of HIF-2α. Numerous studies have established that HIF-2 is the more relevant HIF isoform in terms of RCC tumorigenesis and progression –35. WhilAnother strategy to target HIF-2α is to reduce expression through RNA interference (RNAi). The development of RNAi-based therapies has been challenged by delivery, poor bioavailability due to rapid degradation, poor cellular penetration, and preferential absorption by liver tissue. Arrowhead Therapeutics has developed a next-generation RNAi-based therapy directed against HIF-2α (ARO-HIF2) using a polyconjugate system designed to deliver to the tissue outside the liver. ARO-HIF2 is currently being assessed in a Phase Ib trial in patients with advanced clear cell RCC (NCT04169711).Similar to many other malignancies, clear cell RCC has been characterized by alternations in numerous metabolic pathways . In partMuch effort has been directed towards enhancing the efficacy of immune checkpoint inhibitors in RCC. Preclinical studies have shown that adenosine accumulation in the tumor microenvironment leads to the suppression of the immune activity of cytotoxic T-cell and natural killer (NK) cells and enhancement of the activity of regulatory T-cells (Tregs) and myeloid-derived suppressor cells (MDSC) –46. AdenThe long-established efficacy of high dose (HD) interleukin 2 (IL-2) in a subset of patients with advanced RCC has continued to stoke interest in the potential of cytokine therapy in this disease. Although HD IL-2 has been shown to reliably induce durable and complete remissions in a small group of patients with RCC, this therapy has been limited by its severe toxicity requiring inpatient delivery in specialized centers. Efforts continue to identify predictive models to limit its application to those most likely to benefit. In parallel, pharmaceutical companies have focused on developing novel agents that can both enhance the efficacy of IL-2 and reduce toxicity to allow outpatient administration. Bempegaldesleukin (NKTR-214) is a pegylated CD122 preferential IL-2 agonist designed to stimulate immune cells through the IL2βγ receptor with a limited engagement of the IL2αR subunit to stimulate CD8+ T and NK cells without the accompanying proliferation of Tregs observed with unmodified IL-2. After completing Phase I single-agent assessment, bempegaldesleukin was recently studied in combination with nivolumab in patients with select solid tumors including RCC , 50. Of There are other versions of IL-2 in clinical development as well. ALKS 4230 is a recombinant formulation of IL-2 with intermediate affinity to IL2R, sparing interaction with and simulation of Tregs which are characterized by expression of high-affinity IL2R. ALKS 4230 is currently in clinical assessment in a Phase I trial as a single agent and in combination with pembrolizumab (NCT02799095) . The triIn addition to IL-2, there has been increased interest in other cytokines with similar biologic activity. IL-15 is an immunostimulatory cytokine whose receptor shares the same β and γ chains as the IL-2 receptor but contains a distinct α chain whose engagement results in stimulation of T cells and NK cells and maintenance of memory CD8+ T cells without expansion of T-regs . The devIL-12 is another proinflammatory cytokine that stimulates NK and T-cells to enhance proliferation and IFN-γ production as well as to promote differentiation of T-cells into T helper 1 (Th1) cells . While tBuilding upon the efficacy of the antibodies against PD-1 and CTLA4, a bewildering array of other agents directed against other immune checkpoints have been developed and entered clinical assessment. These include agonistic antibodies against co-stimulatory checkpoint pathways  and blocking antibodies against co-inhibitory checkpoint pathways . While none of these novel checkpoint inhibitors have entered dedicated RCC clinical trials, some preliminary activity in early phase clinical trials has been observed. For example, utomilumab, an agonistic antibody against 4IBB recently demonstrated promising activity in combination with pembrolizumab . Out of The therapeutic bar in advanced clear cell RCC has been raised high in the first-line setting by the recent and expected approvals of multiple combinational regimens. In general, these regimens can reliably produce high response rates and prolonged responses. Nonetheless, complete responses that are durable off-therapy remain rare. Progress in developing new first-line regimens must therefore focus on enhancing the quality of response in terms of depth and durability rather than on incremental increases in response rate or PFS. Towards this end, a greater focus is expected on novel immunotherapies, particularly cytokines. With the vast majority of patients still requiring sequential therapies, there remain significant opportunities to assess novel agents. The novel inhibitors of HIF-2α present the most exciting opportunity to target the central molecular biology of RCC in rational combinations with other agents.Significant improvements have been made in the therapy of patients with advanced clear cell RCC and recently approved combinational regimens have changed the standard of care in the first-line setting. Ongoing Phase III trials will very likely add new combinational regimens to the standard options currently available. Despite this progress, the paucity of complete and durable remissions resulting from treatment with current standard regimens highlights the need to continue to develop novel agents. As we have outlined, the development of novel inhibitors of HIF-2α and glutaminase continues to provide hope that agents targeting the fundamental biopsy of clear cell RCC will improve patient outcomes. In parallel, a multitude of novel immunotherapies seeks to build on the established efficacy of cytokines and checkpoint inhibitors in this disease. While prolonged responses on sequential therapies are in clear progress, the goal remains to cure more patients with advanced clear cell RCC and achievement of this goal requires a commitment to novel therapeutic development.


The coronavirus disease 2019 (COVID-19) vaccination will be the largest vaccination programme in the history of the NHS. Patients on immunosuppressive therapy will be among the earliest to be vaccinated. Some evidence indicates immunosuppressive therapy inhibits humoral response to the influenza, pneumococcal and hepatitis B vaccines. The degree to which this will translate to impaired COVID-19 vaccine responses is unclear. Other evidence suggests withholding MTX for 2 weeks post-vaccination may improve responses. Rituximab has been shown to impair humoral responses for 6 months or longer post-administration. Decisions on withholding or interrupting immunosuppressive therapy around COVID-19 vaccination will need to be made prior to the availability of data on specific COVID-19 vaccine response in these patients. With this in mind, this article outlines the existing data on the effect of antirheumatic therapy on vaccine responses in patients with inflammatory arthritis and formulates a possible pragmatic management strategy for COVID-19 vaccination. Existing work on vaccine response in DMARDs is an imperfect surrogate for COVID-19 vaccine response.MTX may impair humoral response; rituximab likely impairs humoral response for 6 months or longer.Consider risk stratifying rituximab-treated patients and delaying/postponing therapy if appropriate before COVID-19 vaccination.The aim of this viewpoint article is to outline the existing data on the effect of antirheumatic therapy on vaccine responses in patients with inflammatory arthritis, and to formulate a possible pragmatic strategy for the management of therapies in these patients in the context of prospective coronavirus disease 2019 (COVID-19) vaccination. But primarily we aim to facilitate an informed discussion between clinicians and patients in response to issues raised by these data.COVID-19 needs little introduction, and the three effective vaccines produced by Pfizer (mRNA vector), Moderna (mRNA vector) and AstraZeneca (chimpanzee adenovirus vector ChAdOx-1) have provided a potential exit strategy. The COVID-19 vaccine rollout will be the largest mass vaccination programme in the history of the NHS.Vaccinations exert their protective effect by stimulating both humoral and cellular immune responses. The relative importance of humoral and cellular immunity in conferring protection from infection varies with each infective organism . B-cell Immunosuppressive therapy such as the DMARDs, used to treat most of our patients, may impair vaccine responses. Existing data on this topic largely focus on influenza, pneumococcal and tetanus vaccines. There is a small amount of data also available on the Zostavax vaccine. Whether these data can be extrapolated to provide guidance for vaccination strategies in COVID-19 remains uncertain. Patients on immunosuppressive therapy are being prioritized for vaccination, so management decisions will need to be made prior to any additional COVID-19 data being available. Caveats when assessing the literature are noted below in Corticosteroids affect vaccine efficacy in a dose-dependent manner. Several studies have assessed the impact of corticosteroid therapy on humoral response to the pneumococcal and influenza vaccines . Doses >Other than MTX, there is limited evidence for significant impairment of humoral vaccine responses to other conventional synthetic DMARDs (csDMARDs). Sulfasalazine, hydroxychloroquine, azathioprine and leflunomide may reduce vaccine antibody titres but have not been shown to inhibit a seroprotective response to the pneumococcal or influenza vaccines .Much of the trial data on mycophenolate is from organ transplant patients. These trials did not assess responses where mycophenolate was withheld, due to the high risk of graft rejection . MycopheMTX has been shown to impair humoral response to the pneumococcal and influenza vaccines . This isSeveral studies have assessed the impact of anti-TNF therapies on pneumococcal and influenza vaccines. There have been no consistent data linking these treatments to significant impairment of the immune response. However, in patients who are taking concurrent MTX, responses have been shown to be impaired. While seroprotective responses are typically maintained, vaccine antibody titres may be lower than for matched controls , 10, 19.Two large Japanese studies have assessed the impact of IL-6 inhibition on influenza and pneumococcal vaccine responses. One showed impaired responses in the IL-6 inhibitor plus MTX combination treatment arm but no impairment with IL-6 inhibition monotherapy . A subseThere is some conflict within the existing data. Abatacept was shown to impair response to the H1N1 influenza vaccine in comparison to age-matched patients . AbataceJanus kinase (JAK) inhibition may prove to be problematic in the context of the mRNA COVID-19 vaccines, which induce a strong type 1 interferon-driven immune response. Theoretically, inhibition of this pathway could be associated with a diminished response.The effect of baricitinib on pneumococcal conjugate and tetanus toxoid vaccine response was assessed and showed that 68% of patients on long-term baricitinib mounted seroprotective responses to the pneumococcal vaccine, although tetanus toxoid responses were less durable .One study assessed the impact of tofacitinib  on pneumococcal polysaccharide vaccine (PPSV-23) and influenza vaccine response . Here siRecent data in an abstract from the ACR Convergence 2020 has suggested a satisfactory response to the adjuvant herpes subunit zoster vaccine in JAK-inhibitor-treated patients . HoweverB-cell depleting therapy has been shown to impair humoral responses to the influenza and pneumococcal vaccines in several studies and a subsequent meta-analysis , 30, 31.There are some early data suggesting worse outcomes particularly in rituximab treated COVID-19 patients. Case reports have described severe COVID-19 phenotypes in patients treated with rituximab for rheumatological and other B-cell driven disorders . Early sRheumatology departments require guidance on how to manage DMARD/biologic therapies in the context of mass COVID-19 vaccination and this guidance will evolve with time. Existing EULAR guidance is available but may not be sufficient in the context of a global pandemic . In everA summary of the possible challenges specific to rituximab is depicted in For MTX, withholding treatment for 2 weeks following each vaccine dose may help improve humoral response. This is speculative in the context of novel vaccine techniques but could be considered in patients on MTX (and perhaps JAK inhibitors) at low to moderate risk of disease flare. Where a flare occurs, they would require treatment and high doses of prednisolone should be avoided where possible due to its possible effects on vaccine responses and COVID-19 morbidity. However, once again, it is important to stress that the priority is to proceed with vaccination and modification of therapy should not delay this.The situation with JAK inhibitors is unknown, as unlike the MTX study they have only been withheld for 1 week post-vaccination so far. Some data from work on the influenza and PPSV-23 vaccines and the strong type 1 interferon response generated by the mRNA vaccines suggest that withholding JAK inhibitors might improve COVID-19 vaccine responses, but this is speculative. While for abatacept, the data are conflicting and given its mode of action, which could inhibit T-cell responses, treatment guidance urgently requires further evidence.In all cases, any decision to delay treatment should be the result of an informed discussion by each patient and physician on a case-by-case basis.Additional COVID-19-specific data will be critical in producing more evidence-based recommendations. Quantification of COVID-19 vaccine antibody titres, evidence on T-cell immunity and additional work on the impact of booster vaccinations will all be relevant, and there is ongoing work in Leeds collecting such data with and without medication modification. COVID-19 is likely to be a long-term issue, and the data from such a study should be of value for advice on protection and optimal future vaccination strategy.


It is over 4 years since the Prospective Comparison of angiotensin receptor/neprilysin inhibitor (ARNI) with ACEI to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial was published in New England Journal of Medicine. The PARADIGM-HF trial was the one that contributed to the official approval to use ARNI simultaneously with cardiac resynchronisation therapy (CRT) or implantable cardioverter-defibrillator (ICD) in patients who receive optimal medical treatment and still presented NYHA II-IV class symptoms according to the 2016 European Society of Cardiology Guidelines for the diagnosis and treatment of acute and chronic heart failure. The aim of this article is to summarise current knowledge on the activity of ARNI in a selected group of patients with heart failure with reduced ejection fraction (HFrEF) based on a recent PARADIGM-HF subanalysis in the field of renal function in patients with and without chronic kidney disease, glycaemia control in patients with diabetes, ventricular arrhythmias and sudden cardiac death and health-related quality of life. This article includes also recently announced findings on the TRANSITION study which revealed that HFrEF therapy with ARNI might be safely initiated after an acute decompensated heart failure episode, including patients with heart failure de novo and ACEI/ARB naïve, both hospitalised or shortly after discharge, in contrary to the PARADIGM-HF trial, where patients had to be administered a stable dose of an ACEI/ARB equivalent to enalapril 10 mg a day for at least 4 weeks before the screening. It is over 4 years since the Prospective Comparison of ARNI (angiotensin receptor/neprilysin inhibitor—editor’s note) with ACEI (angiotensin-converting enzyme inhibitor—editor’s note) to Determine Impact on Global Mortality and Morbidity in Heart Failure (PARADIGM-HF) trial was published in New England Journal of Medicine. The purpose of the trial was to compare the efficacy of administering enalapril (ACEI) versus sacubitril/valsartan (ARNI) in a selected group of adult patients suffering from heart failure with reduced ejection fraction (HFrEF) of 35% or less and New York Heart Association (NYHA) class II-IV symptoms. The trial was terminated early after a median follow-up of 27 months because of a significantly reduced risk of the primary and secondary endpoint in the sacubitril/valsartan group compared with the enalapril group  Table 11. The PAIn the course of time, subsequent analyses of the PARADIGM-HF trial reveal the ARNI effect to be more and more beneficial for patients with heart failure. The aim of this article is to summarise current knowledge on the influence of ARNI in a selected group of patients with HFrEF, based on a recent PARADIGM-HF subanalysis.Randomised controlled trials have proved that the renin-angiotensin-aldosterone system (RAAS) plays an important role in the pathophysiology of heart failure, thus the morbidity and mortality of patients with HFrEF may be improved by blocking RAAS , 5. In pBrenner et al. showed that RAAS inhibition reduces urinary albumin excretion and slows down the progression to end-stage renal disease, especially in patients without chronic kidney disease (CKD) or diabetes . Treatme2 and 2745 patients of 8399 had CKD; the median urinary albumin/creatinine ratio (UACR) was 1.0 mg/mmol (UACR was available in 1872 patients). The rate of decline in eGFR was significantly lower in the sacubitril/valsartan than in the enalapril arm , despite a greater increase in UACR in the sacubitril/valsartan than in the enalapril arm . The results were similar in patients with and without CKD at screening  was similar between patients with or without CKD at screening, a post hoc analysis of a conventional renal composite outcome  revealed that end-stage renal disease occurred significantly less frequently in patients treated with sacubitril/valsartan, irrespective of presence of CKD at screening. What is more, the increase in UACR was associated with a higher risk of the pre-specified composite renal endpoint only in the enalapril group, but not in the sacubitril/valsartan group.A subanalysis of the PARADIGM-HF trial, performed by Damman et al., concerning the effects of ARNI on renal function in comparison with ACEI, shows superiority of ARNI over ACEI both in patients with and without CKD . At the 2/year  vs − 2.0 ml/min/1.73 m2/year , p < 0.0001) irrespective of the used treatment agent .–33.28–33p < 0.001) compared with enalapril and in deaths due to cardiovascular reasons; the reduction in mortality with sacubitril/valsartan was greater in comparison to enalapril and similar for both sudden cardiac deaths  and deaths due to increasing heart failure .The impact of administration of sacubitril/valsartan on decrease in the number of cardiovascular deaths and all-cause mortality compared with enalapril in patients with chronic heart failure in the PARADIGM-HF trial, is incontestable. Desai et al. examined the data from the trial to better understand the mode of death . AccordiThe precise mechanism of reduced mortality and sudden cardiac death in the group of sacubitril/valsartan remains unclear. However, Sarrias and Bayes-Genis suggest a direct involvement of neprilysin inhibition in the process . Increasvs 15 ± 1.7, p < 0.002), sVT and appropriate ICD shocks , PVCs per hour  and increased biventricular pacing percentage . Such types of original studies provoke questions whether we still need ICDs if we have ARNI , p = 0.15, respectively), with the largest adjusted change score in household chores and sexual relationships  in the group of patients treated with sacubitril/valsartan  in favour of sacubitril/valsartan arm.Similarly, Chandra et al. compared adjusted change scores in most physical and social activities at 8 months and during 36 months . It is sTRANSITION (Comparison of Pre- and Post-discharge Initiation of LCZ696 Therapy in HFrEF Patients After an Acute Decompensation Event) is a randomised, simultaneous, open-label study which aimed at comparing pre- and post-discharge (1–14 days) initiation of sacubitril/valsartan in patients with HFrEF, LVEF ≤ 40% and NYHA class II–IV following hemodynamic stabilisation (defined as no need for intravenous diuretics in 24 h prior to screening and systolic blood pressure > 110 mmHg for at least 6 h prior to randomisation), after an episode of acute decompensated heart failure (ADHF), including patients with newly diagnosed HF and also ACEI/ARB naïve . In contrast, all patients of PARADIGM-HF were administered a stable dose of an ACEI/ARB equivalent to enalapril 10 mg/day for at least 4 weeks before the screening.p = 0.092) [The primary endpoint was the proportion of patients achieving 200 mg sacubitril/valsartan twice a day at 10 weeks’ post-randomisation, and it was reached by 45.0% of 493 patients in the pre-discharge arm and 50.4% of 490 patients in the post-discharge arm  , 43.As the incidence of adverse events of sacubitril/valsartan and their discontinuations was similar in pre- and post-discharge groups, it is concluded that HFrEF therapy with ARNI might be safely initiated after an ADHF episode, including patients with new-onset and ACEI/ARB naïve, both in-hospital or shortly after discharge.The PARADIGM-HF was a well-designed clinical trial that confirmed benefits due to initiation of sacubitril/valsartan in patients diagnosed with HFrEF, LVEF ≤ 35% and NYHA II–IV symptoms despite optimal medical treatment accompanied by cardiac resynchronisation therapy (CRT) or implantable cardioverter-defibrillator (ICD). Although, we do not understand all mechanisms of ARNI activity and the way it decreases HF symptoms, some of the subanalyses provide us invaluable experience on how ARNI reduce the decline in eGFR and risk of hyperkalemia in patients with and without chronic kidney disease, improve glycaemia control in diabetic patients, prevent ventricular arrhythmias or sudden cardiac death and ameliorate health-related quality of life. The new TRANSITION study results, where sacubitril/valsartan is initiated earlier than previously, suggest that ARNI will be used more frequently soon. It brings a hope for patients, affected by HF and who have not responded really positively to a conventional therapy.


B- or T-cell epitopes on the bacterial toxin and to improve the serum half-life of immunotoxins by incorporating an albumin binding domain.Hepatocellular carcinoma (HCC) accounts for most liver cancers and represents one of the deadliest cancers in the world. Despite the global demand for liver cancer treatments, there remain few options available. The U.S. Food and Drug Administration (FDA) recently approved Lumoxiti, a CD22-targeting immunotoxin, as a treatment for patients with hairy cell leukemia. This approval helps to demonstrate the potential role that immunotoxins can play in the cancer therapeutics pipeline. However, concerns have been raised about the use of immunotoxins, including their high immunogenicity and short half-life, in particular for treating solid tumors such as liver cancer. This review provides an overview of recent efforts to develop a glypican-3 (GPC3) targeting immunotoxin for treating HCC, including strategies to deimmunize immunotoxins by removing  Liver cancer remains one of the deadliest cancers in the world despite recent advances in anti-cancer therapeutics . RoughlyHepatocellular carcinoma (HCC) is the most common form of primary liver cancer and is responsible for 75–85% of all reported cases . GeneticPseudomonas exotoxin A, as a treatment for patients with relapsed or refractory hairy cell leukemia that have received at least two prior systemic therapies . . 112]. AAnother strategy for the deimmunization of immunotoxins focused on reducing the T cell response through the elimination of CD4+ T cell activating antigenic peptides ,114,115.In addition to the single deimmunization route, combining the B cell and T cell silencing mutations into a single exotoxin domain has been explored ,117. We The short half-life of recombinant immunotoxins is perhaps the second biggest challenge for immunotoxins in the clinical environment. Pharmacokinetic studies on our HN3-T20 immunotoxin revealed a half-life of around seven minutes in nude mice . It has Streptococcal protein G (ABD) and a second one isolated from a llama VHH antibody (ALB1) to identify which would provide the greatest benefit for our immunotoxin [We tested an albumin binding domain derived from the unotoxin . The addunotoxin . Howeverunotoxin . This inunotoxin . HN3-ALBunotoxin . It is iunotoxin .Our observed improvement of serum half-life was consistent with other reports that showed about a 10-fold increase in half-life with the addition of an albumin binding domain . Our incAnother method for increased serum half-life involves site-directed PEGylation of the immunotoxin . This inPseudomonas exotoxin. We tested the effects of deimmunization strategies and a domain II deletion on enzymatic activity and cancer cell growth inhibition. The HN3-mPE24 and HN3-T20 deimmunized immunotoxins performed the best in our preclinical screen, retaining much of their function. HN3-T20 had the best overall results in vivo, causing the greatest increase in overall survival. Further improvements to the HN3-T20 immunotoxin were tested to improve serum half-life. We compared an ABD domain isolated from Streptococcal protein G and the ALB1 domain isolated from a llama nanobody in our HN3 immunotoxins. The bacterial-derived ABD resulted in greatly improved serum half-life, with low levels of circulating immunotoxin being detected after a 24-hour incubation. With these recent improvements in immunotoxin function, we believe that HN3-based immunotoxins represent an alternative therapeutic option for patients that have not responded to traditional therapies.This review discussed the recent efforts in developing an anti-glypican-3 immunotoxin for clinical use. We have recently shown that the HN3 nanobody displays low off-target staining in complex human tissues and that it directly competes for the Wnt binding site on GPC3. This direct inhibition of Wnt binding leads to a decrease in β-catenin signaling and reduced liver cancer proliferation. To help optimize the toxin domain, we combined HN3 with six different variants of the Clinical trials will provide valuable information about how our deimmunized immunotoxins behave in patients. The point mutations introduced to silence antigenic epitopes are most likely MHC-specific mutations. The large variations in global MHC molecules means that a single deimmunized molecule may work for some individuals and not for others. These same point mutations might in fact increase antigenicity when the MHC background is changed. The complexity of the human immune response most likely means that a universally deimmunized toxin domain is extremely challenging. Gaining a better understanding of how patients from diverse immunological backgrounds respond to immunotoxin therapy would provide important insights. Combining drugs that are involved in immune modulation with immunotoxin treatment may also play a role in the reduction of the immunogenicity of immunotoxins in the clinical setting ,128.Nanobodies represents an ideal platform for the production of bispecific or even trispecific therapeutics ,130,131.It is important to note that the use of our HN3-based immunotoxins may not be limited to the treatment of hepatocellular carcinomas. While the upregulation of glypican-3 has been well established in HCC samples, there is growing evidence that GPC3 may in fact be upregulated in a diverse array of cancers. It has been reported that about half of squamous cell lung cancers ,133,134


Foot health problems are common in the general population, and particularly so in people with rheumatic and musculoskeletal disorders (RMD). Several clinical guidelines state that people with RMDs should have access to foot health services, although service capacity is often limited. The current COVID-19 pandemic has increased the need for alternative ways to provide patient care. The aim of this clinical audit was to review a newly implemented telephone follow-up appointment service conducted within the Rheumatology Podiatry Department in Leeds, UK.Fifty-eight patients attending the Rheumatology Podiatry Department at Leeds Teaching Hospitals NHS Trust were contacted by telephone approximately 6–8 weeks following initial intervention. During the telephone consultation, all patients were asked pre-defined questions relating to their symptoms, intervention efficacy, the need for further appointments and their preference for the type of consultation. To assess the cost of the telephone consultation the number of attempts needed in order to make successful contact, the duration of the call and the number of telephone follow-up appointments completed in a working day were also recorded.Twenty-five patients (43%) were successfully contacted within the 6–8 weeks stipulated time frame and were included in the analysis. Of the 25 contacted, twelve (48%) patients were successfully contacted on the first attempt. Ten (40%) were successfully contacted on the second attempt. The remaining three patients (12%) required 3 or more attempts to make successful contact. Telephone consultations were estimated not to last longer than 10 min, including notes screening and documentation. Eleven patients (44%) reported an improvement in their symptoms, thirteen (52%) reported no change and one patient (4%) reported their symptoms to be worse.Telephone follow-up consultations may be a potentially cost-effective alternative to face-to-face appointments when implemented in a Rheumatology Podiatry Department, and provide an alternative way of providing care, especially when capacity for face-to-face contact is limited. The potential cost saving and efficiency benefits of this service are likely to be enhanced when telephone consultations are pre-arranged with patients.The online version contains supplementary material available at 10.1186/s13047-020-00441-9. Foot health complaints are common worldwide . Garrow In the UK there are generic and disease specific national and local guidelines recommending that people with RMDs and foot problems should have access to foot health services, including the Arthritis and Musculoskeletal Alliance (ARMA) and the UK Podiatry Rheumatic Care Association (PRCA) Standards of Care for People with Musculoskeletal Health Problems , 8. The Advocating the use of telephone and online appointments within various clinical guidelines is reassuring to clinicians during recent times in the wake of COVID-19. Since the declaration of a worldwide pandemic by the World Health Organisation on March 11th, 2020, there has been an unprecedented shift towards alternative methods of consultation within the health care sector. These measures have been implemented in order to minimise the potential for transmission of the virus through non-essential hospital attendance and minimising face-to-face contact between patients and clinicians . Prior tThe aim of this audit was to review a newly implemented telephone follow-up appointment service conducted within the Rheumatology Podiatry Department in Leeds, UK.Patients attending the Rheumatology Podiatry Department at LTHT, Leeds, from January 2015 to May 2015 were included in the audit. Fifty-eight were identified as potential patients. Four of these were identified prospectively while attending the Rheumatology Podiatry Department and 54 retrospectively through screening of the previous eight-week clinic lists. Patients were over the age of 18 at the time of assessment, were under the care of a rheumatologist, and received podiatry interventions such as foot orthoses. Those patients unwilling to participate or unable to understand English were excluded as it was not viable to provide patients with a translator for telephone review at home.The audit was registered with and authorised by the LTHT Quality Improvement Team. As this was not a primary research project, ethical approval was not required.The audit tool consisted of a total of four questions ; mean age 55.5 years (range 25–82). The majority of patients 18/25; 72%) received foot orthoses as the primary intervention for their foot health problem  were contacted successfully on the first attempt; ten (40%) patients required a second or third telephone call before contact was successful; 3 (12%) on the second and 7 (28%) on the third attempt. Two patients (8%) were successfully contacted on the fourth attempt and the remaining 1 (4%) patient, required five attempts  out of 25 patients reported an improvement in their symptoms (GRC of ≥2); eight 32%) receiving foot orthoses, one (4%) receiving debridement/ nail care, one (4%) receiving injection therapy and one (4%) undergoing a medication change. Thirteen (52%) patients reported their symptoms had not changed ; nine (36%) received foot orthoses, three (12%) were managed with exercise prescription and one (4%) received nail care/ debridement. One patient (4%) reported worsening of their symptoms (GRC Score ≤ − 2) after being provided with foot orthoses  were identified prospectively and would have had their telephone review pre-arranged; increasing the likelihood of making successful contact via telephone. All patients were also contacted during what is considered to be normal working hours. These factors could potentially explain why in our community dwelling sample 13 (52%) patients required more than 1 attempt to make successful contact. Taking this into consideration, it could be suggested that in order to make the system run efficiently and effectively, telephone follow-up calls should be pre-arranged in a similar manner to face-to-face appointments. In doing so, this will likely reduce the number of call attempts required before making successful contact.Dudas et al. (2001) restricted the number of attempts to contact patients to three in their study investigating patient satisfaction and outcomes associated with telephone follow-up on discharge from hospital, with those exceeding this number being considered as lost to follow-up . Of the Although data were not formally collected as to the exact duration of each telephone consultation as part of this clinical audit, contacts lasted less than 10 min per patient. This included time needed to obtain and screen medical notes and document the patient’s response to the audit questions , might be those related to reduced non-attendance rates, by patients who do not feel further appointments are necessary and would prefer a short telephone follow-up consultation instead of a face-to-face hospital appointment. There are also broader implications such as reducing the demand on NHS patient transport services often utilised by patients with poor mobility/ additional needs and who require transport to attend face-to-face consultations. The rheumatology podiatry service offered at Chapel Allerton Hospital (LTHT) provides care to patients across the region, with patients often travelling long distances to attend appointments. Following up such patients who express a preference for telephone consultations may reduce patient burden by eliminating the need for potentially unnecessary travel to hospital should intervention be successful, particularly for patients who lack independence or those who are working.At the time of writing, we are currently facing unprecedented challenges in relation to the COVID-19 pandemic with many services traditionally delivered face-to-face health services transitioning to virtual/ telephone consultations in order to minimise hospital visits and physical patient-clinician contact and thus virus transmission . This clTelephone follow-up services have been shown to have no detrimental impact on patient satisfaction –22. HoweThe use of a telephone-based follow-up consultation service within a Rheumatology Podiatry Department may offer a potentially time and cost-efficient alternative to traditional face-to-face consultation appointments for people with RMDs, although the latter will be heavily influenced by local commissioning contracts. Integrating these alongside a traditional approach to service delivery could improve patient throughput by increasing the availability of follow up face-to-face consultation appointments. Adopting an approach such as the one presented within this clinical audit, underpins the need to minimise non-essential face-to-face consultations during the current COVID-19 pandemic. Offering telephone follow-up consultations is likely to be most efficient if they are arranged prospectively to reduce potential losses to follow-up and to improve the efficiency of making successful contact with patients. Research is required to evaluate patient satisfaction of telephone-based follow-up consultations in this clinical setting.Additional file 1.


Plasmids are autonomously replicating genetic elements that are ubiquitous in all taxa and habitats where they constitute an integral part of microbial genomes. The stable inheritance of plasmids depends on their segregation during cell division and their long-term persistence in a host population is thought to largely depend on their impact on the host fitness. Nonetheless, many plasmids found in nature are lacking a clear trait that is advantageous to their host; the determinants of plasmid evolutionary success in the absence of plasmid benefit to the host remain understudied. Here we show that stable plasmid inheritance is an important determinant of plasmid evolutionary success. Borrowing terminology from evolutionary biology of cellular living forms, we hypothesize that Darwinian fitness is key for the plasmid evolutionary success. Performing intracellular plasmid competitions between non-mobile plasmids enables us to compare the evolutionary success of plasmid genotypes within the host, i.e., the plasmid fitness. Intracellular head-to-head competitions between stable and unstable variants of the same model plasmid revealed that the stable plasmid variant has a higher fitness in comparison to the unstable plasmid. Preemptive plasmid competitions reveal that plasmid fitness may depend on the order of plasmid arrival in the host. Competitions between plasmids characterized by similar stability of inheritance reveal plasmid fitness differences depending on the plasmid-encoded trait. Our results further reveal that competing plasmids can be maintained in coexistence following plasmid fusions that maintain unstable plasmid variants over time. Plasmids are not only useful accessory genetic elements to their host but they are also evolving and replicating entities, similarly to cellular living forms. There is a clear link between plasmid genetics and plasmid evolutionary success – hence plasmids are evolving entities whose fitness is quantifiable. Extrachromosomal genetic elements are ubiquitous in prokaryotes and eukaryotes. Viruses, mitochondria, bacteriophages and plasmids are examples for extrachromosomal genetic elements that depend on a hosting cell for reproduction where they replicate independently of the chromosome. Some elements are beneficial for their host , others are characterized as parasitic  or selfish . It is commonly agreed that extrachromosomal elements evolve by ‘descent with modification’ where genetic modifications are heritable over time and generations. Nonetheless, studying the effects of natural selection on the evolution of extrachromosomal elements is a challenging concept; how to disentangle the evolutionary success of extrachromosomal elements from that of their host remains an open question in evolutionary biology.Here we focus on prokaryotic plasmids, which have been extensively studied due to their contribution to gene transfer during microbial evolution. Plasmids encoding for resistance mechanisms to antibiotics confer an immediate advantage to their host fitness under selection for antibiotics resistance. Such plasmids are often found in ecosystems exposed to fluctuating levels of antibiotics e.g., . NonetheLactococcus lactis  that encode multiple functions that support the stable plasmid inheritance or the plasmid persistence in the population e.g., . In contDarwinian fitness is key for the plasmid evolutionary success. Here we define Darwinian fitness of plasmids as the average contribution of a specific plasmid genotype to the plasmid allele pool in the next generation. The fitness of plasmids thus relies on their reproduction, i.e., replication in the cell, and in addition, on their segregation – i.e., inheritance during cell division. We further propose that the concept of plasmid fitness is key for understanding the existence and evolution of plasmids and other extra-chromosomal genetic elements. Notably, natural selection operates on plasmids in two hierarchical levels; one component of plasmid fitness is the host fitness within the population and the second component is the plasmid fitness within the cell. While the first component has been extensively studied, the second component has been so far largely neglected. Here we present a novel approach to compare the fitness of naturally occurring plasmid backbones within the host cell. Our approach includes pairwise in cellulo competitions between selected variants of the same plasmid backbone. We hypothesize that stable plasmid inheritance is an important determinant of plasmid fitness hence a plasmid variant characterized by stable inheritance is expected to outcompete a plasmid variant whose inheritance is unstable.Borrowing terminology from evolutionary research of cellular living forms, we hypothesize that Escherichia coli K12 MG1655. For the comparison of plasmid fitness between plasmid variants, we performed pairwise plasmid competitions between two variants of the same model plasmid in two settings: in the direct ‘head-to-head’ competition, pairs of plasmids are competing upon arrival into the same naïve host cell. In the preemptive competition, an invading plasmid is competed against an endemic plasmid  (Bordetella bronchiseptica (nptII and have a comparable plasmid copy number ranging between 2 and 6 (nptII gene (nptII gene and the oriV region in pCON-S (nptII with the cat gene that confers resistance to chloramphenicol (n = 6), which is comparable to pCON, while pCON-S2 had no measurable loss (n = 6). Both pCON2 and pCON-S2 had no measurable effect on the host fitness  . Our sysiseptica ; the pBBiseptica . The pla 2 and 6 , 2020. W 2 and 6 . The instII gene  that is n pCON-S  . Considering the negligible effect of our model plasmids on the host fitness and the minimal number of host cell divisions in the experiment, the observed differences in plasmid frequencies within the population can be interpreted as evidence for differences in the fitness of individual plasmids within the host. Our approach is parallel to the Luria–Delbrück experiment (termed fluctuation test) that was conceived in order to test for a deviation in the frequency of newly emergent chromosomal mutations from the random expectation  of competing plasmids, we performed direct competitions between the unstable pCON and the stable pCON-S2 as well as the reciprocal set of pCON2 and pCON-S. Plasmid frequency in the population was measured using the proxy of the proportion of hosts harboring the two plasmids. The results of the head-to-head competition between pCON and pCON-S2 reveal that the frequency of the stable pCON-S2 hosts was higher than that of the unstable pCON hosts . The staThe emergence of segregants  was observed in both head-to-head competition experiments. The plasmid loss frequency in the pCON/pCON-S2 competition was quite moderate, affecting 13 (36%) of the populations . In compOverall, the results of the head-to-head competition reveal a significant deviation from the random expectation as the stable plasmid had a higher frequency in the population in comparison to the unstable plasmid across replicates. Consequently, we conclude that the stable plasmid had a higher fitness in comparison to the unstable plasmid. Our results further show that a competition among plasmids in the cell may lead to the exclusion and extinction of unstable plasmids.The establishment of plasmids in a new host may depend on the presence of other resident plasmids in the host . To furtnptII and cat.Taken together, the results of preemptive competition experiments demonstrate that the stable plasmid had a higher fitness in comparison to the unstable plasmid, regardless the order of arrival and the plasmid marker genes Our results so far show that plasmid stability determines plasmid fitness. Nonetheless, plasmids may differ not only with regards to their stability, but also in the traits that they encode. To further examine the effect of plasmid-encoded traits on the plasmid fitness, we compared the fitness of plasmids having similar stability but differ in the antibiotic resistance gene that they carry. For that purpose, we performed pairwise competition experiments between the two stable plasmid variants and between the two unstable plasmid variants.The results of the head-to-head competition between pCON-S and pCON-S2 showed that the frequency of pCON-S hosts was higher than that of pCON-S2 hosts, which were observed in only three replicates in a very low frequency . In addinptII-encoding plasmid over the cat-encoding plasmid despite their equal stability. Whether the expression of nptII or cat has an impact on the host fitness under the conditions we applied in our experiments remain unknown. Our results thus indicate that in a competition between equally stable plasmids, other plasmid properties may be important determinants of the plasmid fitness.Comparing the proportion of pCON-S and pCON-S2 hosts in all competition experiments shows that pCON-S maintained a higher frequency in the population regardless of the order of arrival. The results of the competition experiments between stable plasmids thus show that the presence (or invasion) of a stable plasmid may lead to the exclusion of an equally stable plasmid type. We observe a clear fitness advantage of the The results of the head-to-head competition of both unstable plasmids – pCON and pCON2 – show that half of the replicates included a majority of pCON hosts, while the other half included a majority of host where both plasmids coexisted . The preE. coli MG1655 host. Indeed, we obtained a double resistant clone and subsequent DNA analysis validated that the excised plasmid contained a heteromultimeric plasmid fusion. A diversity of plasmid fusions was reconstituted already after overnight incubation, adding further support that the process of plasmid diversification is rapid. Our results thus demonstrate that plasmid coexistence is achieved by plasmid fusions that are heritable.Our results show that different plasmid variants can be maintained in coexistence. Plasmid coexistence may be explained either by plasmid co-residence in the cell or by plasmid recombination . To further investigate the genetic basis of plasmid coexistence, we analyzed the plasmids from double resistant colonies from each of the “head-to-head” competition experiments. In all of the plasmid hosts we observed large plasmids much above the size of our model plasmids (up to ∼20 Kb). Restriction enzyme digestion analysis showed that these large plasmids corresponded to fusions of both competing plasmids. Hence, the plasmids recombined to form heteromultimeric structures . We did To test the persistence of such plasmid fusions in the population over time, we performed a short-term evolution experiment of representative populations where hosts of both plasmids comprised the majority of the population. For that purpose, we selected three populations from the pCON/pCON-S2 competition experiment . Eight rTo investigate the genetics of the plasmid coexistence in the evolved populations, we analyzed plasmid DNA of representative hosts from eight of the evolved populations (4.1 and 4.3). Again, we observed heteromeric plasmid fusions in all of the analyzed samples, albeit at a much reduced diversity . The decin cellulo plasmid-competitions, we demonstrate the importance of plasmid stability for the evolutionary success  of plasmids, even under very short time scales. Nevertheless, our results reveal that unstable plasmid variants may, in several cases, rise to a high frequency in the population . E. coli DH5α . All primers are listed in MK697350) by insertion of a 254 bp PCR product covering nucleotides 817 to 1044 in pBBR1MCS-5 (GenBank accession no. U25061) into pCON immediately upstream of oriV , was PCR-amplified from plasmid pPCR-Script Cam SK(+) (Agilent technologies), using the primer pair cat-GA-fw and cat-GA-rv. The nptII-less backbone of pCON-S was PCR-amplified using the primer pair pCONSinvFw and pCONSinvRv. Plasmid pCON2 was constructed by replacing the ORF of the nptII gene in pCON with the ORF of the cat gene (see above). Primers used for the PCR amplification of the cat ORF were cat-pCON2-GA-fw and cat-pCON2-GA-rv. The nptII-less backbone of pCON was PCR-amplified using the primer pair pCON2-GA-fw and pCON2-GA-rv.All plasmids in this study were constructed using the Gibson assembly technique , giving in total four sets of competition experiments. Likewise, for each preemptive competition experiment E. coli MG1655 already carrying one of our four model plasmids was transformed with a compatible plasmid in terms of plasmid-encoded marker gene, resulting in three sets of plasmid-plasmid combinations that were reciprocally performed . The four combinations were as follows (endemic versus invading plasmid): pCON versus pCON2, pCON versus pCON-S2, pCON-S versus pCON2, and pCON-S versus pCON-S2. All plasmids were introduced in E. coli MG1655 by electroporation (1 ng per plasmid DNA per electroporation). To increase the transformation efficiency for double transformations (head-to-head competition experiments) concentrations of 4–10 ng per plasmid DNA were used. Electro-competent cells were prepared as reported (E. coli MG1655 host cells. Colonies resistant to both kanamycin (nptII+) and chloramphenicol (cat+) were selected overnight on selective LB plates. Every competition experiment was initiated with six randomly chosen primary double resistant colonies obtained. The six primary transformants were picked and streaked for single colonies on non-selective LB plates. After overnight incubation, for each of the six primary transformants, six single colonies were excised (n = 36 replicates per competition experiment). The colonies were resuspended in 1 ml PBS, serially diluted and aliquots plated on non-selective plates [yielding the total cell number per colony (N)] and plated on kanamycin-supplemented LB plates, chloramphenicol-supplemented plates and plates supplemented with both antibiotics . The proportion of cells carrying only one of the two competing plasmids within one colony were calculated as Pp1 = (NnptII+ - NnptII+cat+)/N for cells carrying either pCON or pCON-S and Pp2 = (Ncat+ - NnptII+cat+)/N for cells for cells carrying either pCON2 or pCON-S2. The proportion of cells carrying both plasmids was calculated as Pp1&2 = NnptII+cat+/N. Plasmid loss was calculated as lossP = 1 - Pp1 - Pp2 - Pp1+2. Our evaluation regime included counting of preferably large and therefore reliable sample sizes for the determination of the different plasmid-carrying cell types. In cases when, due to variability in the plating, colony counts on double selective plates were slightly higher than on plates containing only one of the two antibiotics, counts were corrected toward the counting result on double selective medium in order to avoid negative numerical values in the calculation of the proportions.The success of two plasmid types competed in either the ‘head-to-head’ or preemptive competition mode was determined in single colonies that had grown up non-selectively during 24 h of growth from single cells that initially carried two plasmids. In order to select for double plasmid carriage and to distinguish between the two plasmids, the competing plasmid pairs harbored different marker genes: reported  and plasThe evolution experiment was conducted under non-selective conditions and was founded with double resistant colonies from the preemptive plasmid fitness competition experiment between unstable pCON and stable pCON-S2. In three of the replicates of this experiment  double resistant cells formed the vast majority in the analyzed colonies at the end of the competition. Because of this dominance, double resistant colonies could be readily picked from non-selective plates that were used to estimate the total number of cells within a colony at the end of the competition. For each the replicates, eight colonies were randomly picked and inoculated into 1 ml LB medium for overnight growth at 37°C with constant shaking. At the onset of the experiment samples were plated on double selective media to confirm the double resistant phenotype of the colonies. The overnight cultures were then serially propagated every 24 h in a volume of 1 ml LB medium for three additional transfers using a 1:1000 dilution factor. In total, we recorded ∼64 generations (∼28 generations for the first growth passage on solid medium and ∼9 generations per transfer in liquid medium). Every day, the proportion of plasmid types in the populations was estimated from the proportion of host cells carrying either pCON or pCON-S2 or both as described above for the plasmid competition experiments. At the end of the experiment an individual double resistant colony from four evolved populations of replicate 4.1 and replicate 4.3 were randomly chosen for plasmid isolation followed by diagnostic restriction enzyme digestion.The plasmid loss frequency was estimated from the frequency of plasmid free cells occurring during overnight growth in non-selective media. To determine the loss frequency, cultures were inoculated from single colonies grown on selective media to ensure plasmid carriage. After 12 h growth in 37°C (approximately 8.5 generations), the cultures were serially diluted and plated on non-selective LB media. After overnight incubation, 100 colonies were picked and streaked on selective media (chloramphenicol 20 μg/ml). Colonies grown on selective plates were counted as plasmid carrying. The loss frequency was calculated from plasmid-free cells (not resistant) and the total number of colonies tested.w)  as well as the plasmid pCON have no measurable impact on the fitness of E. coli MG1655  from ovshown in ].TM 1 Kb, Bioline) in 1 × TBE buffer at 4.3 V/cm in 0.7% (w/v) agarose gels containing 1 μg/ml ethidium bromide. Gels were destained with deionized water and documented using the ChemiDoc imaging system (Bio-Rad).Plasmids were extracted from stationary overnight cultures inoculated from single colonies and grown without selection using the GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific). Plasmid DNA was quantified using the Multiskan GO spectrophotometer instrument (Thermo Fisher Scientific). Plasmid conformations were analyzed by restriction enzyme digestions using PstI, ScaI or HindIII following the manufacturers’ guidelines (New England Biolabs). Analytical digestions were electrophoresed against a known standard (Hyperladder© version R2015b.Statistical tests and data analysis were performed in R version 3.5.3  and MatLAll datasets generated for this study are included in the article/NH, TW, and TD designed the experiments. NH, TW, JE, and AG established and performed the experimental work and analyzed the data. TD supported the data analysis. All authors interpreted the results and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.