Gene therapy for pyoderma gangrenosum: optimal transfection conditions and effect of drugs on gene delivery in the HaCaT cell line using cationic liposomes

Background/Aims: Pyoderma gangrenosum (PG) is a rare ulcerative skin disease, currently treated empirically with immunosuppression. PG is a good target for gene therapy since the skin is easily accessible. This study used the FDA-approved vector Lipofectamine® 2000 to investigate in vitro transfection of skin keratinocytes. The aim was to determine an optimum transfection protocol, including the effect of drugs currently used to treat PG on the efficiency of gene transfer, since gene therapy is unlikely to be used as monotherapy. Methods: Cells of the HaCaT line were transfected with the lacZ reporter gene, and transgene expression was measured after a given time period. Conditions tested were: relative concentrations of DNA and Lipofectamine®, time from transfection to measurement of expression, pH, and exposure to clinically relevant drugs (hydrocortisone, methotrexate, infliximab). Results: The greatest levels of β-galactosidase expression were observed using a DNA:Lipofectamine® ratio of 1:5 (μg/μl) on day 3 after transfection, using culture medium at pH 7, and in the presence of hydrocortisone. Transfection efficiency was reduced by the presence of methotrexate and not significantly affected by infliximab. Conclusion: Gene therapy is a potential future strategy for the management of PG; this study is a step towards the development of a topical gene-based agent

Deletion of the protein tyrosine phosphatase PTPN22 for adoptive T cell therapy facilitates CTL effector function but promotes T cell exhaustion

Background Adoptive cell therapy (ACT) is a promising strategy for treating cancer, yet it faces several challenges such as lack of long term protection due to T cell exhaustion induced by chronic TCR stimulation in the tumor microenvironment. One benefit of ACT, however, is that it allows for cellular manipulations, such as deletion of the phosphotyrosine phosphatase non-receptor type 22 (PTPN22), which improves CD8+ T cell anti-tumor efficacy in ACT. We tested whether Ptpn22KO cytolytic T cells (CTL) were also more effective than Ptpn22WT CTL in controlling tumors in scenarios that favor T cell exhaustion. Methods Tumor control by Ptpn22WT and Ptpn22KO CTL was assessed following adoptive transfer of low numbers of CTL to mice with subcutaneously implanted MC38 tumors. Tumor infiltrating lymphocytes were isolated for analysis of effector functions. An in vitro assay was established to compare CTL function in response to acute and chronic re-stimulation with antigen-pulsed tumor cells. The expression of effector and exhaustion-associated proteins by Ptpn22WT and Ptpn22KO T cells was followed over time in vitro and in vivo using the ID8 tumor model. Finally, the effect of PD-1 and TIM-3 blockade on Ptpn22KO CTL tumor control was assessed using monoclonal antibodies and CRISPR/Cas9-mediated knockout. Results Despite having improved effector function at the time of transfer, Ptpn22KO CTL became more exhausted than Ptpn22WT CTL, characterized by more rapid loss of effector functions, and earlier and higher expression of inhibitory receptors (IRs), particularly the terminal exhaustion marker TIM-3. TIM-3 expression, under the control of the transcription factor NFIL3, was induced by IL-2 signaling which was enhanced in Ptpn22KO cells. Anti-tumor responses of Ptpn22KO CTL were improved following PD-1 blockade in vivo, yet knockout or antibody-mediated blockade of TIM-3 did not improve but further impaired tumor control, indicating TIM-3 signaling itself did not drive the diminished function seen in Ptpn22KO CTL. Conclusions This study questions whether TIM-3 plays a role as an IR and highlights that genetic manipulation of T cells for ACT needs to balance short term augmented effector function against the risk of T cell exhaustion in order to achieve longer term protection. What is already known on this topic • T cell exhaustion in the tumor microenvironment is a major factor limiting the potential success of adoptive cell therapy (ACT) in the treatment of solid tumors. • Deletion of the phosphatase PTPN22 in CD8+ T cells improves their response to tumors, but it is not known whether this influences development of exhaustion. What this study adds • Under conditions which promote exhaustion, CTL lacking PTPN22 exhaust more rapidly than WT cells, despite displaying enhanced effector function in their initial response to antigen. • Ptpn22KO CTL express high levels of the inhibitory receptor TIM-3, but TIM-3 signaling does not directly contribute to Ptpn22KO CTL dysfunction. • Ptpn22KO T cells are more responsive to IL-2 through JAK-STAT signaling, which induces TIM-3 expression via the transcription factor NFIL3. How this study might affect research, practice or policy • Strategies aimed at augmenting T cell effector function for ACT should balance improved responses against an increased risk of T cell exhaustion

The role of the smartphone in the transition from medical student to foundation trainee: a qualitative interview and focus group study

Background The transition from medical student to junior doctor is one of the most challenging in medicine, affecting both doctor and patient health. Opportunities to support this transition have arisen from advances in mobile technology and increased smartphone ownership. Methods This qualitative study consisted of six in-depth interviews and two focus groups with Foundation Year 1 Trainees (intern doctors) and final year medical students within the same NHS Trust. A convenience sample of 14 participants was recruited using chain sampling. Interviews and focus groups were recorded, transcribed verbatim, analysed in accordance with thematic analysis and presented below in keeping with the standards for reporting qualitative research. Results Participants represented both high and low intensity users. They used their smartphones to support their prescribing practices, especially antimicrobials through the MicroGuide™ app. Instant messaging, via WhatsApp, contributed to the existing bleep system, allowing coordination of both work and learning opportunities across place and time. Clinical photographs were recognised as being against regulations but there had still been occasions of use despite this. Concerns about public and colleague perceptions were important to both students and doctors, with participants describing various tactics employed to successfully integrate phone use into their practices. Conclusion This study suggests that both final year medical students and foundation trainees use smartphones in everyday practice. Medical schools and healthcare institutions should seek to integrate such use into core curricula/training to enable safe and effective use and further ease the transition to foundation training. We recommend juniors are reminded of the potential risks to patient confidentiality associated with smartphone use

Global burden of 369 diseases and injuries in 204 countries and territories, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019

Background: In an era of shifting global agendas and expanded emphasis on non-communicable diseases and injuries along with communicable diseases, sound evidence on trends by cause at the national level is essential. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) provides a systematic scientific assessment of published, publicly available, and contributed data on incidence, prevalence, and mortality for a mutually exclusive and collectively exhaustive list of diseases and injuries. Methods: GBD estimates incidence, prevalence, mortality, years of life lost (YLLs), years lived with disability (YLDs), and disability-adjusted life-years (DALYs) due to 369 diseases and injuries, for two sexes, and for 204 countries and territories. Input data were extracted from censuses, household surveys, civil registration and vital statistics, disease registries, health service use, air pollution monitors, satellite imaging, disease notifications, and other sources. Cause-specific death rates and cause fractions were calculated using the Cause of Death Ensemble model and spatiotemporal Gaussian process regression. Cause-specific deaths were adjusted to match the total all-cause deaths calculated as part of the GBD population, fertility, and mortality estimates. Deaths were multiplied by standard life expectancy at each age to calculate YLLs. A Bayesian meta-regression modelling tool, DisMod-MR 2.1, was used to ensure consistency between incidence, prevalence, remission, excess mortality, and cause-specific mortality for most causes. Prevalence estimates were multiplied by disability weights for mutually exclusive sequelae of diseases and injuries to calculate YLDs. We considered results in the context of the Socio-demographic Index (SDI), a composite indicator of income per capita, years of schooling, and fertility rate in females younger than 25 years. Uncertainty intervals (UIs) were generated for every metric using the 25th and 975th ordered 1000 draw values of the posterior distribution. Findings: Global health has steadily improved over the past 30 years as measured by age-standardised DALY rates. After taking into account population growth and ageing, the absolute number of DALYs has remained stable. Since 2010, the pace of decline in global age-standardised DALY rates has accelerated in age groups younger than 50 years compared with the 1990–2010 time period, with the greatest annualised rate of decline occurring in the 0–9-year age group. Six infectious diseases were among the top ten causes of DALYs in children younger than 10 years in 2019: lower respiratory infections (ranked second), diarrhoeal diseases (third), malaria (fifth), meningitis (sixth), whooping cough (ninth), and sexually transmitted infections (which, in this age group, is fully accounted for by congenital syphilis; ranked tenth). In adolescents aged 10–24 years, three injury causes were among the top causes of DALYs: road injuries (ranked first), self-harm (third), and interpersonal violence (fifth). Five of the causes that were in the top ten for ages 10–24 years were also in the top ten in the 25–49-year age group: road injuries (ranked first), HIV/AIDS (second), low back pain (fourth), headache disorders (fifth), and depressive disorders (sixth). In 2019, ischaemic heart disease and stroke were the top-ranked causes of DALYs in both the 50–74-year and 75-years-and-older age groups. Since 1990, there has been a marked shift towards a greater proportion of burden due to YLDs from non-communicable diseases and injuries. In 2019, there were 11 countries where non-communicable disease and injury YLDs constituted more than half of all disease burden. Decreases in age-standardised DALY rates have accelerated over the past decade in countries at the lower end of the SDI range, while improvements have started to stagnate or even reverse in countries with higher SDI. Interpretation: As disability becomes an increasingly large component of disease burden and a larger component of health expenditure, greater research and developm nt investment is needed to identify new, more effective intervention strategies. With a rapidly ageing global population, the demands on health services to deal with disabling outcomes, which increase with age, will require policy makers to anticipate these changes. The mix of universal and more geographically specific influences on health reinforces the need for regular reporting on population health in detail and by underlying cause to help decision makers to identify success stories of disease control to emulate, as well as opportunities to improve. Funding: Bill & Melinda Gates Foundation. © 2020 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 licens

Use of FDG PET/CT in identification of bone marrow involvement in diffuse large B cell lymphoma and follicular lymphoma: comparison with iliac crest bone marrow biopsy

Background: Non-Hodgkin's lymphoma (NHL) accounts for around 4% of new cancer cases annually. Bone marrow involvement is important for staging and management. Fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) is used increasingly to identify this, in addition to bone marrow biopsy (BMB), which is seen as "gold" reference standard. Purpose: To compare determination of bone marrow involvement by FDG PET/CT against BMB in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Material and Methods: This was a retrospective study of patients with histologically confirmed NHL at a single UK cancer center undergoing pre-treatment FDG PET/CT and BMB between June 2010 and February 2013. Information was collected from patient notes, cancer registry, histological and imaging reports. Diagnostic accuracy of FDG PET/CT was determined, compared to BMB as the reference standard. Results: Twenty-four patients with DLBCL and 12 with FL were included. Five DLBCL patients had bone marrow involvement on PET/CT; all were confirmed on BMB. Three FL patients had marrow involvement on PET/CT but not on BMB; one FL patient had positive BMB but negative PET/CT. Using BMB as the reference standard, the sensitivity and specificity of FDG PET/CT for detecting bone marrow involvement in DLBCL were 100% and 100%, respectively, and in FL were 0% and 72.7%, respectively. Conclusion: FDG PET/CT is accurate for detection of bone marrow involvement in newly diagnosed DLBCL, but not FL. In DLBCL, positive FDG PET/CT may negate the need for routine BMB, although BMB in addition or combination may be appropriate if this would influence management or prognosis

Report from Working Group 3: Beyond the Standard Model physics at the HL-LHC and HE-LHC

This is the third out of five chapters of the final report [1] of the Workshop on Physics at HL-LHC, and perspectives on HE-LHC [2]. It is devoted to the study of the potential, in the search for Beyond the Standard Model (BSM) physics, of the High Luminosity (HL) phase of the LHC, defined as $3$ ab$^{-1}$ of data taken at a centre-of-mass energy of 14 TeV, and of a possible future upgrade, the High Energy (HE) LHC, defined as $15$ ab$^{-1}$ of data at a centre-of-mass energy of 27 TeV. We consider a large variety of new physics models, both in a simplified model fashion and in a more model-dependent one. A long list of contributions from the theory and experimental (ATLAS, CMS, LHCb) communities have been collected and merged together to give a complete, wide, and consistent view of future prospects for BSM physics at the considered colliders. On top of the usual standard candles, such as supersymmetric simplified models and resonances, considered for the evaluation of future collider potentials, this report contains results on dark matter and dark sectors, long lived particles, leptoquarks, sterile neutrinos, axion-like particles, heavy scalars, vector-like quarks, and more. Particular attention is placed, especially in the study of the HL-LHC prospects, to the detector upgrades, the assessment of the future systematic uncertainties, and new experimental techniques. The general conclusion is that the HL-LHC, on top of allowing to extend the present LHC mass and coupling reach by $20-50\%$ on most new physics scenarios, will also be able to constrain, and potentially discover, new physics that is presently unconstrained. Moreover, compared to the HL-LHC, the reach in most observables will, generally more than double at the HE-LHC, which may represent a good candidate future facility for a final test of TeV-scale new physics

Report from Working Group 3 : Beyond the Standard Model Physics at the HL-LHC and HE-LHC

CERN Yellow Reports: Monographs, vol 7 (2019)Contribution to: HL/HE-LHC WorkshopThis is the third out of five chapters of the final report [1] of the Workshop on Physics at HL-LHC, and perspectives on HE-LHC [2]. It is devoted to the study of the potential, in the search for Beyond the Standard Model (BSM) physics, of the High Luminosity (HL) phase of the LHC, defined as $3$ ab$^{-1}$ of data taken at a centre-of-mass energy of 14 TeV, and of a possible future upgrade, the High Energy (HE) LHC, defined as $15$ ab$^{-1}$ of data at a centre-of-mass energy of 27 TeV. We consider a large variety of new physics models, both in a simplified model fashion and in a more model-dependent one. A long list of contributions from the theory and experimental (ATLAS, CMS, LHCb) communities have been collected and merged together to give a complete, wide, and consistent view of future prospects for BSM physics at the considered colliders. On top of the usual standard candles, such as supersymmetric simplified models and resonances, considered for the evaluation of future collider potentials, this report contains results on dark matter and dark sectors, long lived particles, leptoquarks, sterile neutrinos, axion-like particles, heavy scalars, vector-like quarks, and more. Particular attention is placed, especially in the study of the HL-LHC prospects, to the detector upgrades, the assessment of the future systematic uncertainties, and new experimental techniques. The general conclusion is that the HL-LHC, on top of allowing to extend the present LHC mass and coupling reach by $20-50\%$ on most new physics scenarios, will also be able to constrain, and potentially discover, new physics that is presently unconstrained. Moreover, compared to the HL-LHC, the reach in most observables will, generally more than double at the HE-LHC, which may represent a good candidate future facility for a final test of TeV-scale new physics