Improvements in laparoscopic partial nephrectomy (LPN) in order to minimize perioperative warm ischemia time (WIT), complications, and consequently patient outcome are desirable. Veriset™ is a ready-to-use hemostatic patch of absorbable oxidized cellulose and hydrogel components that has earlier been implemented in vascular and hepatic surgery. We report our experience using this device in LPN.n = 40) or conventional suture technique (n = 40). Patient characteristics, operation time and WIT, postoperative course and complications were recorded retrospectively. Tumor complexity was calculated according to the R.E.N.A.L. score. Outcome was determined according to the “trifecta” criteria .Patients with a solitary malignant renal mass suspicious for renal cancer underwent LPN with either the use of Veriset™ hemostatic patch (p = 0.001) and WIT were both lower in the Veriset™ group . No differences in surgical margins (p = 0.602) and overall complication rates at 30 (p = 0.599) and 90 days (p = 0.611) postoperatively were noticed. The surgical outcome according to “trifecta” was achieved in 65% of patients using Veriset™ and in 57.5% of patients by suture closure, respectively.No significant differences with regard to clinical parameters and median R.E.N.A.L. score (6) were observed between both groups. Operation time (mean 127.1 min vs. 162. 8 min; The hemostatic Veriset™ patch can successfully be implemented in LPN. Handling and application appear favorable, thereby reducing operation time and WIT. The present results suggest that the device may represent an alternative to parenchyma suturing in LPN. Approximately 99,200 new cases of renal cell carcinoma (RCC) are diagnosed per year and RCC is expected to account for almost 39,100 cancer-related deaths in the European Union in 2018 . While tA total of 85 consecutive patients with suspicious renal mass on computed tomography (CT) or magnetic resonance imaging (MRI) underwent LPN from April 2016 to September 2018 at the Department of Urology, University Hospital Tuebingen. Five patients were excluded: two cases were converted to laparoscopic radical nephrectomy; two cases were converted to an open approach due to massive adhesions; one patient got an additional adrenalectomy. All tumors were single lesions. After tumor excision, in 40 cases a conventional parenchyma suturing was performed (suture group). In the other 40 cases, a Veriset™ patch was applied to the parenchyma defect without additional parenchyma suturing (Veriset™ group).Patient characteristics and surgical parameters , pathological characteristics and postsurgical outcomes  were retrospectively recorded from the institutional cancer database. Tumor complexity was evaluated according to the R.E.N.A.L. nephrometry score. Complications within 30 and 90 days after surgery according to the modified Clavien classification  were docLPN was performed by a transperitoneal approach by experienced laparoscopic surgeons. Three ports  were placed when the lesion was on the left side. An additional 5 mm port was used on the right side to retract the liver. The 10 mm camera trocar was inserted pararectally via a mini-laparotomy and the intraabdominal pressure was adjusted to 12–15 mmHg. The retroperitoneum was exposed by mobilization of the hemicolon and the renal hilus was initially explored and the renal vessels identified and isolated. Hereafter, the kidney was completely mobilized and the tumor was exposed. Warm ischemia was performed by clamping of the renal artery with an endo-bulldog clamp. Afterwards, the tumor was sharply dissected from the surrounding renal parenchyma. If the collecting system was opened, it was closed with a 4-0 polyglactin suture. Selection of parenchyma closure via suture or sealing device was based on preference of the surgeon. For the conventional parenchyma closure, single knot endo-clip polyglactin sutures according to the technique initially described by Lahodny were performed . In the Unclamping was performed after completion of the suture or application of the patch, respectively. In both approaches, the Gerota fascia was reestablished. Finally, the tumor was removed in an endobag.t-test for continuous variables. χ2 and Mann–Whitney U tests were used to compare categorical variables with p ≤ 0.05 considered statistically significant. Statistical analyses were performed using commercial software .Statistical analysis was performed using Students’ n = 80 patients. The median age of the patients was 62 years, 58.7% of patients (n = 47) were men and 41.3% (n = 33) women. No significant difference in subgroup composure with regard to clinical parameters was noted. Detailed patient characteristics are shown in Table p = 0.113). Overall, 67.5% of renal tumors identified as malignant in the Veriset™ group and 65% in the suture group. A positive surgical margin was detected in one patient each in both cohorts (Table The study cohort comprised p = 0.001), respectively. In both subgroups, three patients were operated under zero ischemia. The WIT was 14.6 min in the Veriset™ group and 20.6 min in the conventional suture group (p = 0.01). The collecting system was opened and sutured in 35% (n = 14) of patients in the Veriset™ group and in 27.5% (n = 11) of patients in the suture group.The mean operative time accounted for 127.1 min in the Veriset™ group and 162.8 min in the suture group (n = 32) of patients in the Veriset™ group and 67.5% (n = 27) in the suture group (p = 0.209) and no difference in postoperative drop of hemoglobin level was noted (p = 0.426).Stable GFR > 90% of preoperative level was observed in 80% (n = 11) in the Veriset™ group and 20% (n = 8) in the suture group at 30 days (p = 0.599) postoperatively. The 90-day complication rates were 30%  and 22.5% , respectively (p = 0.611). Further sub-analysis after 30 days revealed two grade III  and one grade IV  complication in the Veriset™ group and two grade III (urinary extravasation: n = 2) and one grade IV  complication in the suture cohort. At 90-day follow-up, in both groups one additional grade III complication was recorded (Table n = 26) of patients in the Veriset™ group and 57.5% of patients (n = 23) in the suture group (p = 0.494). Adding renal function preservation as defined by stable GFR to “trifecta”, the rate of optimal surgical outcome was 57.5% (Veriset™ group: n = 23) and 37.5% (suture group: n = 15), respectively (p = 0.025).Overall complication rates were 27.5% (Sutures for the closing of renal parenchyma defects during laparoscopic and robotic-assisted PN represent a standard technique and various approaches have been proposed . HoweverVeriset™ is a ready-to-use hemostatic patch designed for endoscopic and open surgery. The patch contains of oxidized cellulose and is impregnated with buffer salts, trilysine and a reactive polyethylene glycol and does not contain human or animal coagulation factors . For itsRegarding postoperative complications and outcomes, in the present analysis, no differences in overall complication rates and the optimal outcome according to “trifecta” criteria were detected using the Veriset™ device. However, when renal function preservation was combined with “trifecta” outcomes, a significant benefit for the Veriset™ technique could be detected. Noteworthy, in both patient subgroups three grade III complications were observed. In the Veriset™ group, two patients developed pseudoaneurysm and one patient had secondary hemorrhage. With regard to postoperative risk of bleeding, it should be reflected that Veriset™ serves as a mechanical hemostat by concentrating and activating platelets and clotting factors for hemostasis. The patch is not biologically active itself and is considered as passive hemostat. In conclusion, for adequate function of the patch, the patients’ own intact coagulation system is a prerequisite . NoteworThe fact that postoperative pseudoaneurysms were noted in the Veriset™ patient subgroup is another finding that should be closely monitored during the further evaluation of the device. Interestingly, Shigeta et al. proposed that the combined application of a TachoSil™ patch and renorrhaphy led to a reduced incidence of pseudoaneurysms as compared to parenchyma sutures only . Other hIn general, application of the patch during laparoscopy was favorable, and no damage of the patch itself or loss of the functional layer after passage through the port was noted. For follow-up studies, Veriset™ owns favorable characteristics, since due to its complete absorption, it should not mimic tumor recurrence in imaging studies.Finally, there are several limitations looking at the present analysis. First, the design of the study being retrospective and non-randomization of comparison subgroups are limitations that must be acknowledged. Inherent bias due to the fact of surgeons’ preference for the selection of type of parenchyma sealing cannot be excluded, despite no differences in tumor characteristics in the comparative groups. Moreover, the number of cases is overall still limited. Further analysis in randomized trials for LPN appears however warranted, given the favorable results from the present exploratory analysis. Overall, in both groups the mean tumor diameter was small, which should be reflected with regard to the application in larger RCC.Moreover, only patients undergoing conventional laparoscopy were included. Given the growing caseload of robotic-assisted PN , it appeIn conclusion, the hemostatic Veriset™ patch can successfully be implemented in LPN. Handling and application appear favorable and the present results indicate time-preserving properties with regard to overall operation time and warm ischemia time. The patch may therefore be an alternative to parenchyma suturing in LPN that warrants further systematic evaluation.


To quantify the association between type 2 diabetes remission and 5‐year incidence of cardiovascular disease outcomes, overall and in pre‐defined subgroups.1c level was assessed.Retrospective cohort analysis of 60,287 adults with type 2 diabetes from the Care and Health Information Analytics (CHIA) database. Multivariable Cox models were used to assess the association between remission within the first two years of follow‐up and incidence of cardiovascular disease (CVD) outcomes including events, microvascular and macrovascular complications at 7‐year follow‐up. Effect modification by age, sex, diabetes duration, pre‐existing CVD, baseline body mass index (BMI) and HbA7489 (12.4%) people achieved remission during the first two years of follow‐up. Overall, remission was associated with lower risk of CVD outcomes. Remission was associated with lower risk of microvascular complications for younger compared with older age groups (eg aHR: 0.59 (0.41–0.84) and aHR: 0.78 (0.67–0.92) for those aged <45 years and 75–84 years, respectively). Amongst those achieving remission, those with no or 1–2 comorbidities had lower risk of microvascular complications (aHR: 0.65 (0.56–0.75)) compared to those with more than three comorbidities (aHR: 0.83 (0.69–0.99), respectively). There were no significant interactions in the remaining subgroups or for models assessing CVD events or macrovascular complications.Achieving remission of type 2 diabetes is associated with a lower risk of microvascular complications, particularly for younger groups and those with fewer comorbidities. Targeted interventions that focus on promoting remission in these groups may reduce the impact of microvascular complications and associated health costs. Remission of type 2 diabetes has been shown to be achievable through lifestyle changes alone. The extent to which remission is associated with reduced cardiovascular disease (CVD) outcomes in key subgroups of people with type 2 diabetes has not previously been assessed. We found that achieving remission of type 2 diabetes was associated with a lower risk of microvascular complications, particularly for younger groups and those with no or few comorbidities. Targeted interventions that focus on promoting remission in these groups may reduce the impact of microvascular complications and associated health costs. It could be valuable to understand determinants that predict CVD events in people who achieve remission to allow interventions to be targeted and personalised. In the present study, we therefore sought to examine the remission‐CVD link overall and in pre‐defined subgroups using a large population‐based cohort with established type 2 diabetes from routine clinical care.22.1We used data from the Care and Health Information Analytics database (CHIA), a pseudo‐anonymised live electronic database linking routinely collected primary care data for approximately 1.5 million people from 150 general practitioner (GP) practices across Hampshire and Isle of Wight  with clinical biochemistry data from local hospital laboratories. We identified a cohort of 60,715 adults (aged 18–85 years) over 7 years who were coded for type 2 diabetes based on the Quality and Outcomes Framework (QOF) Read code criteria prior to 1 January 2013, and who had continuously recorded electronic records over seven years from the 1 January 2013 to 1 April 2020.2.21c level <48 mmol/mol (6.5%) measurements separated by a minimum period of 6 months in the absence of diabetes medications  or bariatric surgery.Remission (at any point during the first two years of the study period: 1 January 2013–2031 December 2015) was defined as two consecutive HbA2.31c), total cholesterol, HDL‐cholesterol, eGFR).Baseline data on sociodemographic characteristics such as age, sex and ethnicity , socio‐economic status (defined using the 2019 Index of Multiple Deprivation (IMD) quintiles, a small‐area measure of socio‐economic status, ranked nationally and comprises seven domains: income, employment, education/skills/training, health and disability, crime, barriers to housing and services, and living environment) were available. Clinical variables at baseline included the following: comorbidities , asthma, cancer, dementia, atrial fibrillation, epilepsy, heart failure, stroke, peripheral vascular disease, hypertension, osteoporosis, osteoarthritis and depression), frailty (defined using the electronic frailty index score), latest smoking status, weight and biochemistry measures , amputation and stroke), microvascular complications  and macrovascular complications , peripheral arterial disease (PAD) or amputation) occurring between years 2 and 7 of follow‐up. QoF definitions for each diagnosis were used.2.5Missing data on biochemistry variables, remission status, baseline weight and IMD were assumed to be missing at random and multiply imputed using chained equations using STATA SE 16.0. Ten cycles of imputation were used. All imputed data after patient death were recoded as missing. Missing data on ethnicity were recoded as white ethnicity.2), normal (18.5–24.9kg/m2), overweight (25–29.9kg/m2), obese (≥30kg/m2)],1c  Descriptive statistics were used to compare baseline sociodemographic and clinical characteristics for individuals who did and those who did not achieve remission during the first 2 years of follow‐up. Cox proportional hazards models were fitted to estimate the association between remission in the first 2 years of follow‐up and 5‐year incidence of CVD events, macrovascular complications and microvascular complications. These models included only those alive at the end of the first 2 years of follow‐up. The mid‐point of the quarter of death was used as exact date of death was not available. Multivariable models were adjusted based on a priori reasoning for age, sex, ethnicity, IMD, pre‐existing CVD, BMI, diabetes duration, number of comorbidities and clustering within practices . We additionally adjusted for last observed BMI and hypertension status. We then modelled interaction terms between remission and age , sex , diabetes duration , pre‐existing CVD , amputation, and stroke), baseline BMI [underweight (<18.5kg/m33.11c data at baseline and at least one follow‐up measurement). The cohort was followed for a mean of 6.8 (SD = 1.3) years. Table The study cohort included 60,287 people with type 2 diabetes for whom remission status could be assessed (ie had HbA3.2Three thousand four hundred and eighteen (5.7%), 12121 (20.1%) and 4839 (8.0%) people had a CVD event, microvascular or macrovascular complication during the last 5 years of follow‐up. Compared with those who did not, those who achieved remission had a significantly lower risk of CVD (aHR: 0.75 (0.64–0.88)), microvascular complications (aHR: 0.68(0.62–0.74)) or macrovascular complications (aHR: 0.78 (0.69–0.89)) at 7 years follow‐up. Univariate‐ and multivariate‐adjusted associations are presented in Table 3.31c level at baseline did not modify the association between remission and microvascular complications. Similar results were observed amongst those who did not have pre‐existing CVD, microvascular or macrovascular complications at baseline.Table 41c level did not modify the remission‐CVD association.In this large population‐based cohort of 60,287 people with type 2 diabetes, we found that remission was associated with a substantially lower risk of CVD outcomes including events, microvascular and macrovascular complications. The effect on microvascular complications was modified by age and number of comorbidities. Younger age and fewer comorbidities amongst those achieving remission were associated with a lower risk of microvascular complications. Sex, diabetes duration, pre‐existing CVD, BMI and HbAThis study extends the literature by examining the association between remission (in the absence of medication or surgery) of type 2 diabetes and CVD outcomes. Although the literature around glucose control and CVD outcomes  is mixed,1c and cardiovascular events in a Dutch population.In contrast with some previous studies, we do not find evidence of effect modification by pre‐existing CVD.1c measurements over the seven‐year duration of the study period. Limitations include the presence of missing data on variables which may reflect under‐testing or under‐reporting in routine data. Although we were able to use multiple imputation techniques which provide valid statistical inferences under the missing at random assumption, it is not possible to test for this assumption. Our data on medication were limited to specific periods of time (6‐month blocks), which may have misclassified some individuals’ medication exposure and therefore remission status. However, given the large size of the cohort, it is likely that any effect would be small. Exact date of death was also not available in the dataset which may have resulted in under or overestimation in time to event analyses. As with most studies using routine data, we were not able to distinguish between intentional or unintentional remission and did not have data on cause of death. It is possible that some of our findings may be confounded by unintentional remission, though some studies have found this to have minimal impact on outcomes.Strengths of the study include the use of a large observational cohort with extended follow‐up, which allowed interaction effects to be examined, as well as the availability of data on comorbidities, medication and all HbAGiven the limited NHS resources and growing prevalence of type 2 diabetes, prioritising subgroups for targeted and personalised interventions is challenging. Our findings suggest that a focus on younger age groups and those with fewer comorbidities could be one approach with potential to reduce the impact of microvascular complications, as they are more likely to benefit most. Targeted interventions that focus on promoting remission as an achievable outcome in these groups could be justified given the potential to reduce the burden of microvascular complications and associated costs.None to declare.Hilda Hounkpatin: Conceptualization‐Equal, Formal analysis‐Lead, Investigation‐Lead, Methodology‐Lead, Writing‐original draft‐Lead, Writing‐review & editing‐Equal. Beth Stuart: Funding acquisition‐Equal, Methodology‐Supporting, Writing‐review & editing‐Equal. Andrew Farmer: Conceptualization‐Equal, Funding acquisition‐Equal, Writing‐review & editing‐Equal. Hajira Dambha‐Miller: Conceptualization‐Lead, Funding acquisition‐Lead, Methodology‐Equal, Writing‐review & editing‐Equal.CHIA is an anonymous National Health Service database, and all individuals have consented for collection of their medical records for inclusion in the database. Ethical and governance approval for this study was obtained from the University of Southampton (ERGO 56127), and Care and Health Information Exchange Information Governance Group (CHIE IGG).


Manufacturing has been the key factor limiting rollout of vaccination during the COVID‐19 pandemic, requiring rapid development and large‐scale implementation of novel manufacturing technologies. ChAdOx1 nCoV‐19  is an efficacious vaccine against SARS‐CoV‐2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large‐scale production. We discuss the process economics and the “distributed manufacturing” approach we have taken to provide the vaccine at globally‐relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID‐19 vaccine supply at low cost. Well over a billion doses of the ChAdOx1 nCoV‐19 vaccine developed by The University of Oxford and AstraZeneca have been produced and distributed at low cost. This represents a substantial proportion of global COVID‐19 vaccine supply to date, especially in low‐ and middle‐income countries. The authors report on the development of the manufacturing process for the vaccine, and the process economics and distributed manufacturing approach involving bulk vaccine (drug substance) production on five continents. To our knowledge the greatest previously‐disclosed scale of manufacture of adenovirus vectors for human use was the production of c. 2 × 1017 VP (for 2.7 million doses) of Janssen Vaccines' adenovirus‐vectored Ebola vaccine  , followed by a multistep downstream process  . In the second quarter of 2020 we introduced process modifications, removing bottlenecks to enable commercial manufacturing at 1000–4000 L scale. The first 1000 L drug substance batch was completed in September 2020, and the billionth dose was released in July 2021.22.1The ChAdOx1 nCoV‐19, ChAdOx1 Lassa‐GP, ChAdOx1 luciferase, and ChAdOx2 GFP vectors used here have previously been described , with a working volume of 25–35 ml in a 125 ml flask unless otherwise stated. BioBlu 3c and 14c (Eppendorf) single‐use bioreactor vessels were used in accordance with the manufacturers' instructions. A GX bioreactor controller unit and C‐BIO software  were used to control both vessel types. Dissolved oxygen (DO) was regulated at a setpoint of 55% air saturation by addition of medical air via macrosparger. pH was regulated in the range 7.2–7.3 as previously described  was used in 50 and 200 L STRs. 0.05 culture volumes of BalanCD feed was added when the density reached 1.0 × 106 culture cells/ml. At a cell density of 4.0 × 106/ml (range 3.0–6.0 × 106), cells were diluted with 1 volume of medium and infected, using an MOI of 10 unless otherwise stated. 0.05 volumes of BalanCD feed were added 30 min after infection, and again after 22 h.50 and 200 L upstream processes were performed using Pall Allegro™ stirred tank reactors (STRs). For work using the high MOI fed batch process  were added to cell culture at 48 ± 4 and 96 ± 4 h post bioreactor inoculation. Bioreactor temperature was reduced to a lower set point within 4 h of the second feed. About 140 h postinoculation, the cell culture was harvested and processed for analysis.For the low MOI upstream process Figure , bioreac2.3Lysis was performed as previously described . Column equilibration was in accordance with the manufacturer's instructions. After loading, capsule was washed with 10 membrane volumes (MV) of equilibration buffer before the elution step (both at 5 MV/min).For “direct‐load” AEX (loading clarified lysate), the small‐scale studies shown in Figure For the “direct‐load” AEX purifications from a 10 L bioreactor Figure  a perist2, 5% w/v sucrose. After loading, the capsule was washed with 10 MV of equilibration buffer followed by at least 20 MV of wash buffer . Product was eluted with 5 MV of elution buffer . Loading, equilibration, washing, and elution were all performed at 2.5MV/min.For the “direct‐load” AEX purification from a 1000 L bioreactor Figure  a PK50 lSmall‐scale studies shown in Figure 2.6Product quantification was as previously reported, using quantitative polymerase chain reaction (qPCR) and UV spectrophotometry assays for viral particles in impure and pure samples, respectively, and an immunostaining‐based infectivity assay  was quantified using the HEK293 HCP ELISA kit (Cygnus Technologies), according to the manufacturer's instructions. Residual host cell DNA was quantified using a previously reported quantitative PCR method targeting a 94 base pair amplicon within the Alu repeats . A detailed execution protocol for drug substance production  was combined with mass balance data as described in this manuscript to build and parametrize a model. Assumptions made were designed to be conservative: productivity of 2 × 1033.17 cells/ml with high viability  per ml, around fivefold greater than typically obtained in our previous USP , cell density and time of harvest Figures . Althougs Figure .We have previously demonstrated and discussed advantages of the use of a cell line/promoter combination which represses antigen expression during production  remained in the range 2–4 × 1011 VP/ml, with acceptable cell growth and metabolite profiles  of a high MOI species E adenovirus production process is relatively low. Considering the high particle:infectivity ratio of these vectors , after estimating likely losses in processing and aliquoting and adding a margin for safety, we felt we could only rely on production of c. 300 IU of useable seed per cell infected during seed production: this would be sufficient to infect only 30 cells at an MOI of 10. This 30‐fold amplification factor implied the need for large numbers of batches of working virus seed (creating a testing burden and consistency concerns), and a possible requirement for multi‐step seed expansion (from master virus seed through two working seed production passages). Reduction of the MOI to c. 3 was considered, but we felt this would be unlikely to fully address these concerns.To avoid the complexity arising from a high seed requirement, we investigated use of MOI < 1. By infecting cells at low density within 24 h of bioreactor inoculation with cells, and then continuing culture for a further 120 h, we allowed the initially‐uninfected cells to proliferate before becoming secondarily infected by the released virus particles from first round of infection Figure . InformeThe reduced MOI, and resulting increased virus amplification factor, has enabled seed from a single 200 L bioreactor batch to supply global manufacturing needs.3.32 per 40 L of lysate) and although we anticipated this could be reduced with optimization, we were concerned that TFF membrane supply could become problematic due to high demand during the COVID‐19 pandemic.The initial TFF step was identified as the key bottleneck for DSP scale‐up. Time did not permit extensive process characterization before scale‐up, and we were concerned that excessive concentration of the lysate during TFF would increase risk of formation of aggregates of product with host‐cell protein and DNA. Using only modest concentration to favor process robustness meant that multiple process volumes of diafiltration buffer and waste needed to be handled, which we anticipated would be challenging in some facilities. The original process also required large TFF membrane areas , was likely to prove limiting for the speed of global COVID‐19 vaccination rollout. We were also concerned that so‐called vaccine nationalism was likely to impede equitable access to effective vaccines, as had been seen during the H1N1 swine flu pandemic Fidler, .We therefore set out to design a global manufacturing strategy to achieve three goals: low cost; speed to large volume supply, including prompt wide geographical availability; and, critically, consistent high quality.Low cost was achieved due to the high productivity of the process and the use of off‐the‐shelf equipment and materials. Modeling of DS manufacturing using a commercial cost‐modeling package suggests a cost of goods of <EUR 1 per dose Figure . This foDespite the productivity of the process, it was apparent multiple manufacturing facilities were required to achieve speed to large volume supply: global supply required >1 m litres of upstream process output and hence a large number of batches. Moreover, location of DS manufacturing appeared likely to be a key determinant of vaccine availability, especially for low and middle income countries. From the outset (Q1 2020), our strategy therefore included simultaneous technology transfer to multiple sites, including in India and China. Through Q2‐3 2020, this strategy was further developed, resulting in distribution of drug substance manufacturing across a network of existing contract manufacturing and licensee sites capable of single‐use bioreactor (SUB) viral manufacturing at 1000–4000 L scale Figure .To achieve the earliest possible release of commercial product through this distributed manufacturing strategy, we “parallel‐tracked” a number of process development, tech transfer, validation and regulatory activities which would more typically run sequentially Figure . For sev4Vaccine supply is proving to be the key determinant of timing of COVID‐19 vaccination, especially in low and middle‐income countries.Some aspects of adenovirus‐vectored vaccine manufacturing are inherently well‐suited to pandemic response. Antigen‐repressing promoter/cell combinations, and the absence of the encoded antigen from the adenovirus virion, enable similar upstream, downstream and drug product manufacturing regardless of the encoded antigen, and we had previously described a small‐scale process designed for rapid response to emerging pathogens  facilitates the implementation of the process using standard equipment available at most sites, reduced material requirements, and achieved chromatography cycling times as short as 2 h at 2000 L scale. Efficient recovery and high quality of adenovirus from such a “direct load” AEX has not to our knowledge previously been reported.10 VP) per litre of upstream culture. We believe this productivity is around double that of previously disclosed batch or fed batch processes for adenovirus manufacture . Combined with the biosafety of the vector (BSL1‐2 dependent upon jurisdiction), the design of the process enabled a distributed manufacturing strategy, with several independent national or regional supply chains. Such strategies have previously been proposed as means of ensuring wide geographical availability of vaccines, but never to our knowledge implemented with this speed and scale. In the context of current calls for waivers of intellectual property relating to COVID‐19 vaccines, it is important to note the degree of central co‐ordination which has been necessary to ensure efficient manufacture and consistent quality across the multiple production sites Figure .The process described here has thus provided a substantial proportion of global COVID‐19 vaccine supply to date (>1 billion doses released for supply as of August 2021). Uniquely among current COVID‐19 vaccines from high‐income country developers, the programme has aimed to achieve “equity by design”: as a result of the process development and technology transfer strategy, the majority of output has been manufactured and/or used in low/middle income countries.C. C. D. J., Y. L., S. F., G. G., A. B., R. R. S., Te. L., S. C. G., A. J. R., and A. D. D. are named inventors or contributors to intellectual property assigned to Oxford University Innovation relating to the ChAdOx1 nCoV‐19 vaccine and/or manufacturing process, and may receive a proportion of proceeds from out‐licensing of the intellectual property. J. J., Th. L., N. B., D. P., R. V., and R. T. are employees of AstraZeneca.Conceptualization: Alexander D. Douglas and Richard Turner. Experimental design, planning, execution, and data acquisition: Carina C. D. Joe, Jinlin Jiang, Teresa Lambe, Yuanyuan Li, Sofiya Fedosyuk, Gaurav Gupta, Adam Berg, Rameswara R. Segireddy, David Mainwaring, Amar Joshi, Paul Cashen, Byron Rees, Jonathan Humphreys, Sarah Davies, Nick Smith, Scott Bruce, Dennis Verbart, Daan Bormans, Carol Knevelman, Mark Woodyer, Lee Davies, Lisa Cooper, Maria Kapanidou, Nicole Bleckwenn, Daniel Pappas, Daniel C. Smith, Catherine M. Green, Raghavan Venkat, Adam J. Ritchie, Richard Turner, and Alexander D. Douglas. Construction of process model: Nitin Chopra and Piergiuseppe Nestola. Provision of critical materials: Teresa Lambe and Sarah C. Gilbert. Funding acquisition: Sarah C. Gilbert, Richard Turner, and Alexander D. Douglas. Writing and review of manuscript: all authors.Supporting information.Click here for additional data file.


The contribution of Th17 cytokines to autoimmune thyroid disease (AITD) is generally accepted. However, the roles of Th17 cells in the initiation and progression of Hashimoto's thyroiditis (HT) and Graves' disease (GD) remain unclear. Selenium deficiency, along with genetic predisposition and environmental factors, may have a role in thyroid autoimmunity.We aimed to assess (1) the Th17 immune response by measuring plasma levels of Th17‐ and Treg‐associated cytokines and (2) the selenium status in treatment‐naïve Latvian patients with newly diagnosed GD or HT.Eleven GD patients, 41 HT patients, and 26 healthy subjects were recruited for this study. Plasma levels of IL‐17a, IL‐22, IL‐23, IL‐6, and IL‐10 were detected by xMAP technology, while selenium was detected fluorometrically.No significant differences in IL‐17a, IL‐22, IL‐23, IL‐6, or IL‐10 levels were found among the HT patients, GD patients, and controls. In the HT patients, IL‐17a levels were positively correlated with IL‐22, IL‐23, IL‐6, and IL‐10, while IL‐22 was correlated with IL‐6, IL‐23, and IL‐10. In the GD patients, IL‐17a levels were positively correlated with IL‐22, IL‐23, and IL‐10; IL‐22 was positively correlated with IL‐23, IL‐6, and IL‐10; FT3 was positively correlated with IL‐17a, IL‐23, and IL‐10; and FT4 was positively correlated with IL‐17a and IL‐10 levels. Plasma selenium levels were negatively correlated with antithyroid peroxidase antibody titers in the HT patients. Although no difference in selenium levels was observed between the AITD patients and controls, the selenium status of the Latvian patients with GD or HT was at a suboptimal level. Th17 cytokines were positively correlated with the severity of hyperthyroidism, suggesting their possible role in Graves' disease pathogenesis. Plasma selenium levels were negatively correlated with antithyroid peroxidase antibody titers in the Hashimoto's thyroiditis patients In both autoimmune disorders, the complex interactions among various environmental and endogenous factors trigger thyroid autoimmunity in predisposed individuals, leading to enhanced thyroid autoantigen presentation and impairment of immune tolerance.The understanding of the Th1 and Th2 cell dichotomy, which is essential for autoimmune disorders, shifted with the discovery of Th17 lymphocytes. Supporting evidence suggesting the contribution of Th17 cells and pro‐inflammatory IL‐17 to AITD, particularly HT, has been produced in the last decade.Currently, a role for selenium deficiency in the development of AITD is suggested along with genetic predisposition and environmental triggers. Selenium deficiency can impair the differentiation of CD4+ T cells, leading to cellular and humoural response dysfunction. Selenium leads to changes in the levels of IL‐6, TGF‐β, and IL‐23 and may further contribute to impaired differentiation of Th17 cells.In this study, we aimed to assess (1) the Th17 immune response in AITD by measuring the plasma concentrations of Th17‐ and Treg‐associated cytokines and (2) the selenium status in Latvian patients with newly diagnosed, treatment‐naïve GD or HT to determine whether the selenium level is reduced in AITD patients compared to healthy subjects without autoimmune disorders.22.1p = .139). The diagnosis of AITD was confirmed by evaluating the clinical manifestations of GD and HT, complemented by biochemical thyroid tests and ultrasound imaging. The control group consisted of 26 age‐ and sex‐matched healthy subjects without any autoimmune disease and with normal thyroid function, who were negative for anti‐thyroid peroxidase autoantibodies (anti‐TPO), anti‐thyroglobulin autoantibodies (anti‐Tg), antinuclear antibodies (ANAs), and anti‐tissue‐transglutaminase IgA (tTG‐IgA) autoantibodies.Eleven adult patients with untreated newly diagnosed hyperthyroid GD and 41 patients with untreated new‐onset HT were recruited into this prospective study between January and November 2020 at the Department of Endocrinology, Riga East University Hospital, Latvia. Thirty‐three out of 41 HT patients were euthyroid , and 8 out of the 41 patients had mild subclinical hypothyroidism . The median age was 36 (27–50.5) years for the HT patients, 41 (29–62) years for the GD patients, and 30 (27–42.25) years for the control group (The exclusion criteria for the study included (1) pregnancy; (2) presence of malignancy, active infection, chronic inflammatory, or other autoimmune disease; (3) use of selenium‐containing commercial supplements; (4) vegetarian or vegan diet; (5) use of medications affecting thyroid and immune function ; and (6) significant renal or hepatic impairment.The study protocol was conducted according to the Declaration of Helsinki and approved by the Riga East Hospital Research Committee and the Central Medical Ethics Committee, Latvia (Decision No. 01‐29.1/5033). All patients and volunteers provided written informed consent before enrollment and blood sample collection.2.2Peripheral blood samples were collected from all 86 patients and volunteers after overnight fasting early in the morning. Thereafter, the samples were centrifuged at 1500 rpm for 10 min and stored at −80°C until analysis. Venous blood samples were collected from hyperthyroid and hypothyroid patients before therapy with methimazole or levothyroxine, respectively.2.3The levels of serum‐free thyroxine (FT4), free triiodothyronine (FT3), and thyroid‐stimulating hormone (TSH), as well as anti‐TPO and anti‐Tg were measured by chemiluminescence immunoassay (Siemens) performed on an Advia Centaur XP (Siemens) analyzer. The normal values were as follows: FT4, 0.7–1.48 ng/dl; FT3, 0.2–0.44 ng/dl; and TSH, 0.35–4.94 μIU/ml; anti‐TPO, 0‐5.61 IU/ml; anti‐Tg, 0‐40 U/ml. The levels of anti‐TSHR, ANAs, and tTG‐IgA autoantibodies were measured by ELISA (Pharmacia Diagnostics Freiburg) according to the manufacturer's instructions .2.4Cytokine patterns analyzed in the current study included the following groups: Th17‐related cytokines (IL‐17a and IL‐22), Th17‐promoting cytokines (IL‐23 and IL‐6), and a Treg‐associated cytokine (IL‐10). EDTA plasma immunological markers were detected by xMAP technology . All tests were performed in accordance with the manufacturer's instructions . The limit of detection for each cytokine was as follows: IL‐17a, 0.009 pg/ml; IL‐22, 0.021 ng/ml; IL‐23, 0.098 ng/ml; IL‐6, 1.7 pg/ml; and IL‐10, 0.3 pg/ml.2.5The plasma selenium concentration was determined fluorometrically by using the fluorescence spectrophotometer “Cary Eclipse” .2.6H test followed by the two‐stage step‐up method of Benjamini, Krieger, and Yekutieli as a post hoc procedure, and the results are displayed as the median and interquartile range. Depending on the data distribution, both parametric Pearson's analysis and nonparametric Spearman's correlation analysis were performed to determine the relationships of studied parameters. p < .05 was considered statistically significant for all statistical tests. All graphical images and statistical analyses were performed using GraphPad Prism 9.0 for MacOS software (GraphPad Software).The normal distribution of data was confirmed by D'Agostino and Pearson, Anderson–Darling, and Shapiro–Wilk normality tests. Homogeneity of variances was tested using Brown–Forsythe and Bartlett's tests. As dispersion did not correspond to a normal distribution in most cases, data were analyzed by the nonparametric Kruska–Wallis 3n = 33) and hypothyroid  patients. No significant differences in IL‐17a, IL‐22, IL‐23, IL‐6, or IL‐10 levels were found among the patients with HT, patients with GD and controls  . The plasma levels of cytokines are summarized in Table The demographic and biochemical characteristics of the participants are presented in Table p values representing the association level between studied cytokines in the patient groups are given in Figure When the relationships among Th17‐related cytokines were investigated, we found that in HT patients, the plasma levels of IL‐17a were positively correlated with the levels of IL‐22, IL‐23, IL‐6, and IL‐10 Figure ; additiop > .05). However, in newly diagnosed GD patients, the levels of FT3 were positively correlated with IL‐17, IL‐23, and IL‐10 .Moreover, no significant correlations were found between the levels of interleukins and thyroid autoantibodies, TSH, FT3 or FT4 in HT patients  .The median plasma selenium levels were 85.03 (60.09–116.60) µg/L for HT patients, 71.33 (59.05–104.42) µg/L for GD patients, and 80.35 (67.15–113.67) µg/L for control participants Table . Similarp > .05). The levels of selenium failed to correlate with other characteristics, such as age, titers of anti‐Tg and anti‐TSHR, TSH, FT3, and FT4 .When the associations between plasma selenium and cytokines were analyzed, no significant correlations were found between selenium levels and the plasma levels of IL‐17a, IL‐22, IL‐23, IL‐6, and IL‐10 for all groups  cells. We failed to demonstrate any differences in IL‐22 levels among the three groups of subjects enrolled in this study. However, Ruggeri et al.IL‐22 produced by Th17 cells can also be secreted by another novel subset of Th cells—CCRIn this study, positive correlations between the levels of IL‐22, IL‐17a, IL‐23, and thyroid hormones were found in newly diagnosed GD patients. However, no correlations between the levels of interleukins and thyroid‐specific autoantibodies were found. These results suggest that Th17 cytokines may have a stimulatory effect on the severity of hyperthyroidism independent of autoantibody levels. No such correlation was found in the HT group as a vast majority of the recruited HT patients presented as euthyroid. Due to the limited availability of treatment‐naïve patients, only a small number of GD patients was recruited. Therefore, the results of GD group analyses must be concluded and interpreted with caution because of the small sample size in this group.Among the nongenetic factors involved in the pathogenesis of thyroid autoimmunity, high iodine intake, and a low selenium status appear to be the most important. Previous studies have suggested the immunomodulatory and protective role of selenium and selenoproteins. It has been suggested that selenium deficiency presents with inhibition of selenoproteins and altered secretion of IL‐10, IL‐12p40, and IFN‐γ, leading to Th1/Th2 imbalance by shifting the balance toward Th1 responses.In a population‐based Danish study, Pedersen et al.Various plasma or whole‐blood selenium cut‐offs have been proposed by different authors to reflect selenium sufficiency. The activity of GPx3 in human serum is used as a marker of selenium sufficiency. It has been shown that the plasma selenium level needed to optimize platelet GPx activity is 100–115 μg/L, as proposed by Alfthan et al.,5Despite the recent data on the involvement of the IL‐17/IL‐23 axis in the development of thyroid autoimmunity available in the literature, we did not find any significant differences in the plasma levels of Th17‐associated cytokines between patients with AITD and control subjects. However, a positive correlation between Th17 ILs was detected in both HT patients and GD patients. Furthermore, Th17 cytokines were positively correlated with the severity of hyperthyroidism, suggesting their possible role in GD pathogenesis. A few limitations should be considered when interpreting our data. Although simultaneous assessment of various cytokines provided more information on the complex cytokine pattern that characterizes AITD, a small number of AITD patients may lead to a possible loss of statistical significance. Due to the lack of data regarding the levels of other Treg cytokines, it will be of interest to further pursue studies of Th17 and Treg cells as important players in AITD pathogenesis. Although no difference in selenium levels was observed between AITD patients and controls, the selenium status of Latvian patients with newly diagnosed GD or HT was at a suboptimal level. Interestingly, plasma selenium levels were negatively correlated with anti‐TPO autoantibody titers in HT patients. Moreover, HT patients with higher anti‐TPO levels had lower levels of selenium, suggesting that these patients might benefit from selenium supplementation.Ilze Konrade and Tatjana Zake designed the study protocol, Tatjana Zake, Sabine Upmale‐Engela, Ieva Kalere, Gita Gersone, Andrejs Skesters collected the data and performed patient selection, Gita Gersone and Andrejs Skesters performed laboratory work, Tatjana Zake, Ieva Kalere, Sabine Upmale‐Engela, Gita Gersone, Simons Svirskis, Andrejs Skesters analyzed and interpreted the data, Tatjana Zake, Ieva Kalere, Valerija Groma, Ilze Konrade, Simons Svirskis wrote and edited the manuscript, Ilze Konrade and Valerija Groma supervised the study. All authors read and approved the final manuscript.The authors declare that there are no conflict of interests.


To determine the biological significance of zonal origins in prostate cancer.Altogether, 270 consecutive radical prostatectomy cases from 2009 to 2012 were adopted. Cases were divided into those having transition zone (TZ) cancer or peripheral zone (PZ) cancer. Cases with indeterminate tumor location and central zone cancers were excluded from the analyses. Prognosis and clinicopathological features were compared between the two tumor locations. Biochemical recurrence (BCR) and clinical progression (CP) were adopted as prognostic outcome measures. Immunohistochemical features of the v‐ets avian erythroblastosis virus E26 oncogene homolog (ERG)/serine peptidase inhibitor, Kazal‐type 1 (SPINK1) status, and loss of phosphatase and tensin homolog (PTEN‐loss), as well as conventional preoperative and postoperative characteristics, were analyzed.This cohort comprised 93 cases of TZ cancer and 160 cases of PZ cancer. TZ cancer cases showed significantly higher BCR and CP‐free survival rate than PZ cancer cases. Notably, no TZ cancer cases developed CP during the 7.8 years of median follow‐up time. Tumor location was an independent predictive factor for BCR in the multivariate analysis. Additionally, TZ cancer cases showed a significantly lower prevalence of ERG‐overexpression and PTEN‐loss than PZ cancer cases .TZ cancer cases showed a better prognosis and different immunohistochemical features. Conservative treatment strategies could be considered for TZ cancer cases. Significant variations have been reported for the incidence of PCa between races and regions. For instance, Asian countries are known to have a low incidence, which is one‐tenth compared to the highest incidence in European countries,McNeal proposed the anatomical classification of the prostate in the late 1960s, which has been widely used for clinicopathological studies of the prostate.Although the genetic features of PCa remain unclear to date, the racial differences associated with the most frequent genetic alterations of the transmembrane protease, serine 2 (TMPRSS2)‐v‐ets avian erythroblastosis virus E26 oncogene homolog (ERG) gene fusion and phosphatase and tensin homolog (PTEN) inactivation have been reported. The prevalence of the TMPRSS2‐ERG gene fusion/ERG‐overexpression in PCa was approximately 50% in Western countries,Recent advances in PCa diagnosis, such as multiparametric magnetic resonance imaging (MRI) and MRI‐ultrasound fusion targeted biopsy, have enabled clinicians to diagnose PCa location without surgery. Here, the knowledge of the biological features of PCa of different zonal origins is crucial for the daily practice of PCa management, especially for selecting treatment options such as focal therapies. Previous reports including our research have shown that clinicopathological differences exist between TZ and PZ cancers. In these studies, TZ cancer cases showed a lower grade and earlier stage than PZ cancer cases.22.1Altogether, 270 consecutive radical prostatectomy (RP) cases diagnosed by a single genitourinary pathologist (HT) from August 2009 to August 2012 were adopted. Cases with any preoperative treatment, such as endocrine therapy, radiation therapy, and chemotherapy, were excluded. Eighteen cases with indeterminate cancer location or CZ location were excluded from statistical analyses as mentioned below. Hence, 252 cases were ultimately analyzed.2.2The RP specimens were routinely processed, and pathological diagnosis was made as previously reported.In the RP specimens, intraductal carcinoma was examined by one genitourinary pathologist (SS) according to the McNeal's criteria.Immunohistochemical analyses for anti‐ERG rabbit monoclonal antibody , anti‐PTEN rabbit monoclonal antibody , and anti‐SPINK1 mouse monoclonal antibody  were performed using BenchMark XT automated stainer . In the immunohistochemical analyses, the cut‐off value was set as 10%. Thus, cases were categorized as ERG/SPINK1 positive or PTEN‐loss if 10% or more of tumor cells stained for ERG/SPINK1 antibody or lost the staining for PTEN antibody.2.3For each patient, clinical information such as age, serum PSA level, clinical T stage, and postoperative follow‐up data, were collected from medical records.2.4We adopted biochemical recurrence (BCR) and clinical progression (CP) as the primary and secondary measures of treatment outcome, respectively. The definition of BCR was as previously described.2.5In the first analysis, we compared the clinicopathological features and prognoses between TZ and PZ cancers. The Mann‐Whitney U‐test was performed for comparing continuous  variables, and the Fisher's exact test was used for categorical variables. BCR‐free and CP‐free survival rates were calculated by the Kaplan–Meier method, and the inter‐group difference was compared by the log‐rank test. Additionally, multivariate Cox regression analysis for BCR was performed to examine the prognostic value of tumor location. Different multivariate models were assigned for preoperative and postoperative features  was used for the analyses.In all the analyses, statistical significance was defined as a 2.6This study received prior approval from the Ethics Committee of the affiliated institution . Informed consent was not required due to the retrospective study design.33.1P = .587).This cohort comprised 93 cases (34.4%) of TZ cancer and 159 cases (58.9%) of PZ cancer. The clinicopathological patient backgrounds are shown in Tables P = .186). Moreover, no significant difference was shown for tumour volume and positive surgical margin rate. The immunohistochemical profile in the entire cohort was as follows: ERG‐overexpression shown in 14.8%, PTEN‐loss in 13.0%, and SPINK1 expression in 15.6%. Compared to PZ cancer cases, TZ cancer cases showed an extremely lower prevalence of ERG‐overexpression (3.2% vs 20.1%) and PTEN‐loss (2.2% vs 18.2%) with a significant difference . A significant difference was not shown for SPINK1 expression (P = .858).Regarding postoperative pathological findings, TZ cancer cases showed significantly lower GG and pT than PZ cancer cases. Lymph node metastasis was detected more frequently in PZ cancer cases (8.8%) than in TZ cancer cases (3.2%), although a significant difference was not shown (3.2P = .854). TZ cancer cases showed a significantly higher BCR‐free survival rate than PZ cancer cases , as well as PSA, cT, and GG in biopsy (Table P = .036), as well as GG in RP specimen, positive surgical margin, and lymph node metastasis (Table P = .0021), although it was not an independent predictive factor in the multivariate analysis (P = .2585). The ERG/SPINK1 status was not predictive of BCR in both the univariate and multivariate analyses in the entire cohort.The median follow‐up period was 93.8 (interquartile range: 59.3‐102) months in the entire cohort, 93.4 (60‐101.8) months in TZ cancer cases, and 93.6 (55.3‐101.9) months in PZ cancer cases without a significant difference . The prevalence of PTEN‐loss, which was associated with a lower BCR‐free survival rate, was significantly higher in the PZ cancer cases. These results of PTEN status suggest that the difference in the prevalence of PTEN‐loss caused prognostic differences between two tumor locations. Several studies have shown the lower prevalence of TMPRSS2/ERG gene fusion/ERG‐overexpression in TZ cancer cases.Here, we examined the immunohistochemical features of TZ and PZ cancers using three antibodies, ERG, PTEN, and SPINK1. The TZ cancer cases showed a significantly lower prevalence of ERG‐overexpression and PTEN‐loss. However, the prevalence of SPINK1 expression did not significantly differ according to the two tumor locations. Regarding the relationship between ERG/SPINK1 expression and clinicopathological parameters, among TZ cancers, ERG‐/SPINK + cases showed a significantly lower BCR‐free survival rate than ERG‐/SPINK1‐ cases. In contrast, among the PZ cancer cases, ERG‐/SPINK1‐ cases showed the highest GG (We showed a variety of clinicopathological differences between TZ and PZ cancers, including novel findings in the prognosis of CP and immunohistochemical features. These findings will provide important implications for diagnosing and treating PCa. The TZ cancers were shown to be associated with a lower detection sensitivity by Prostate Imaging‐Reporting and Data System, Version 2,In conclusion, TZ cancers showed better outcomes and different clinicopathological features than PZ cancers. Thus, TZ cancers may be treated more conservatively.None declared.


The mechanism underlying carcinogenesis and the genomic features of superficial non‐ampullary duodenal epithelial tumors (SNADETs) have not been elucidated in detail. In this study, we examined the genomic features of incipient SNADETs, such as small lesions resected via endoscopic treatment, using next‐generation sequencing (NGS).Twenty consecutive patients who underwent endoscopic treatment for SNADETs of less than 20 mm between January and December 2017 were enrolled. Targeted genomic sequencing was performed through NGS using a panel of 160 cancer‐related genes. Furthermore, the alteration/mutation frequencies in SNADETs were examined.The maximum size of the SNADETs examined in this study was 12 mm in diameter. Five SNADETs were classified as low‐grade dysplasia (LGD) tumors, while 14 SNADETs were classified as high‐grade dysplasia tumors. Only one carcinoma in situ was detected. NGS data for 16 samples were obtained. APC alterations were detected in 81% of samples (13/16). KRAS, BRAF, and TP53 alterations were detected in 25% (4/16), 18.8% (3/16), and 6.3% (1/16) of cases, respectively.APC alterations in most small SNADETs resected via endoscopic treatment, from LGD to carcinoma samples. Even in SNADETs classified as small LGD exhibited KRAS and BRAF alterations.We detected  APC alterations were detected in most small superficial non‐ampullary duodenal epithelial tumors (SNADETs) resected via endoscopic treatment. Even in SNADETs classified as small low‐grade dysplasia, KRAS and BRAF alterations were present in a few samples. Extensive studies of associations between gene alterations in key driver genes and CRC metastasisKRAS, TP53, SMAD4, and BRAF. Similar mechanisms to those in CRC, such as the adenoma‐carcinoma sequence, may contribute to the pathogenesis of duodenal adenocarcinoma.APC, KRAS, and BRAF alterations.In colorectal cancer (CRC), the adenoma‐carcinoma sequence describes the process of carcinogenesis.In this study, we examined the genomic features of incipient SNADETs, such as small lesions resected by endoscopic treatment, using next‐generation sequencing (NGS).Twenty consecutive patients (20 samples) who underwent endoscopic treatment for SNADETs less than 20 mm in diameter between January and December 2017 at Hokkaido University Hospital were enrolled. Cold snare polypectomy (CSP) and endoscopic mucosal resection (EMR) are generally indicated for lesions that are ≤10 mm and ≤20 mm in diameter,This study was approved by the institutional review board of Hokkaido University Hospital . Written informed consent was obtained from each participant. All experiments were performed in accordance with the ethical guidelines of the 2013 Declaration of Helsinki.All resected specimens were routinely fixed in 10% buffered formalin for 24 h at room temperature. Thereafter, the specimens were serially sliced at a width of approximately 2 mm and embedded in paraffin following routine methods. All sections were cut to a thickness of 3 μm and stained with hematoxylin and eosin for microscopic examination. Paired peripheral blood samples were collected from each patient and stored at −80°C.Clinicopathological findings were reviewed, including age, sex, tumor location, tumor color, tumor size, tumor macroscopic type, resection method, histological type, and phenotype of the resected specimen. Macroscopic typing of SNADETs was based on the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma.et al.Immunohistochemical staining was performed using the dextran polymer‐peroxidase‐based EnVision System  and metal‐3,3′‐diaminobenzidine . Finally, sections were counterstained with Mayer's hematoxylin. Membrane staining for CD10  and cytoplasmic staining for MUC2  and MUC5AC  were judged as positive when >5% of tumor cells showed a positive reaction for each marker. Based on CD10 expression and mucin phenotypes (MUC2 and MUC5AC) determined by immunoreactivity, SNADETs were further subclassified into five groups according to the criteria proposed by Yao Each resected specimen was sectioned into five slices (8‐μm‐thick slices), and macroscopic trimming was performed to obtain as many cancer cells as possible for more than 50% tumor cellularity. Genomic DNA was extracted from formalin‐fixed paraffin‐embedded (FFPE) tissue samples using a GeneRead DNA FFPE Kit  according to the manufacturer's instructions. Genomic DNA was extracted from the blood samples using a genomic DNA extraction kit . The concentration and purity of genomic DNA samples were determined using a NanoDrop system  and Qubit dsDNA HS Assay Kit (Life Technologies) designed to be accurate for sample concentrations of 10–100 ng/mL. Genomic DNAs from the FFPE tissue and blood samples were stored at −80°C until analysis.Multiplex PCR was performed using a GeneReadDNAseq Panel PCR Kit V2 (Qiagen) and Human Comprehensive Cancer Panel (Qiagen), which included 160 cancer‐related genes. Finally, an optimized library was constructed using a Gene Read DNA Library I Core Kit (Qiagen). The library was analyzed using an Agilent DNA 1000 Kit Bioanalyzer . Library preparation was achieved within two working days. The enriched libraries were sequenced to obtain paired‐end reads (2 × 150 bp) using the MiSeq NGS platform , resulting in a mean depth of >500×. The sequencing data were analyzed using an original bioinformatics pipeline, GenomeJack, tuned for clinical sequence examination, “CLUHRC” .t‐test. Differences were considered statistically significant when P < 0.05.The results were analyzed using Prism version 6 . Data are expressed as means ± standard errors of the mean. Parameters were compared between two groups by Fisher's exact test or Student's This study included 20 consecutive SNADETs resected by endoscopic treatment Table . The maxAPC alterations were detected in 81%  of cases. KRAS, BRAF, and TP53 alterations were detected in 25% , 18.8% , and 6.3%  of cases, respectively . Ultimately, we analyzed 16 libraries through NGS. There were no copy‐number variations. APC alterations in the LGD  and HGD/CIS groups , KRAS alterations in the LGD  and HGD/CIS groups , BRAF alterations in the LGD  and HGD/CIS groups , or TP53 alterations in the LGD  and HGD/CIS groups  . There were no significant differences between the rates of APC alterations in SNADETs (i.e. 81%). Additionally, there were no significant differences between the rates of APC alterations in the LGD group (80%) and the HGD/CIS group (81.9%). Kojima et al. reported an APC alteration frequency of 54.5% in duodenal adenoma.APC plays a critical role in CRC development as a tumor suppressor gene, and its gene product inhibits Wnt/β‐catenin signaling.et al.APC knockout. These findings suggest that upregulation of the Wnt/β‐catenin pathway is a major factor in the initial stages of duodenal adenoma/adenocarcinoma carcinogenesis. Our results further support the key role of APC in duodenal adenomas/adenocarcinomas.We observed a high frequency of BRAF and KRAS alterations typically arise at the adenoma stage of the adenoma‐carcinoma sequence,APC alteration. KRAS and BRAF encode proteins belong to the Ras–Raf–MEK–ERK signaling pathway. The activation of this pathway is considered a molecular switch, leading to cell growth and proliferation.KRAS and BRAF are associated with a risk of developing advanced neoplasiaKRAS, BRAF, and TP53 alterations were detected in 25, 18.8, and 6.3% of patients, respectively. Surprisingly, we detected KRAS and BRAF alterations in 40% (2/5) and 20% (1/5) of LGD lesions, respectively. These findings are consistent with a previous study showing that one in five cases of LGD (20%) harbor a KRAS alteration.KRAS or BRAF alterations and those without alterations within wild‐type sequences.In CRC, TP53 is a key driver gene in CRC progression and is frequently detected in small bowel advanced adenocarcinoma.TP53 alteration. These results support the hypothesis that the accumulation of genetic alterations after an initial APC might cause progression from adenoma to carcinoma in SNADETs. Considering our results and those of previous reports,KRAS or BRAF alterations, which might result in progression to HGD or carcinoma.This study had several limitations. It included a relatively limited number of samples (20 samples) and did not include submucosal invasive cancer samples. There were no lesions with a gastric phenotype in the collected samples. Additionally, we performed genome sequencing analysis using the Human Comprehensive Cancer Panel (Qiagen), which included 160 cancer‐related genes. Therefore, we could not analyze other gene alterations and epigenomic changes in SNADETs. These limitations should be considered when interpreting the study results. Therefore, further studies that include a larger number of cases and lesions with a gastric phenotype are needed in the near future.APC alterations in most SNADETs from LGD to carcinoma samples. Even in SNADETs classified as small LGD (<12 mm in diameter), KRAS and BRAF alterations were present in few samples.In conclusion, in the incipient SNADETs, such as small lesions resected by endoscopic treatment, we detected


Acidiphilium multivorum and Leptospidillum ferriphilum, isolated from acid mine drainages. The consortium was inoculated in a dissolution of e‐scraps powder and cultured for 15 days. Forty‐five elements were analyzed in the liquid phase over time, including silver, gold, and 15 REEs. The bioleaching efficiencies of the consortium were >99% for Cu, Co, Al, and Zn, 53% for Cd, and around 10% for Cr and Li on Day 7. The second step consisted of a microalgae‐mediated uptake from e‐scraps leachate. The strains used were two acidophilic extremotolerant microalgae, Euglena sp. (EugVP) and Chlamydomonas sp. (ChlSG) strains, isolated from the same extreme environment. Up to 7.3, 4.1, 1.3, and 0.7 µg by wet biomass (WB) of Zn, Al, Cu, and Mn, respectively, were uptaken by ChlSG biomass in 12 days, presenting higher efficiency than EugVP. Concerning REEs, ChlSG biouptake 14.9, 20.3, 13.7, 8.3 ng of Gd, Pr, Ce, La per WB. Meanwhile, EugVP captured 1.1, 1.5, 1.4, and 7.5, respectively. This paper shows the potential of a microbial sequential process to revalorize e‐scraps and recover metals and REEs, harnessing extremotolerant microorganisms.Electronic scraps (e‐scraps) represent an attractive raw material to mine demanded metals, as well as rare earth elements (REEs). A sequential microbial‐mediated process developed in two steps was examined to recover multiple elements. First, we made use of an acidophilic bacteria consortium, mainly composed of  The article shows the potential of a microbial sequential process to revalorize electronic scraps and recover metals and rare earth elements harnessing extremotolerant microorganisms. There are already examples of the harnessing of scraps, such as the regeneration of lithium electrode scraps to obtain new cathodes for lithium‐ion batteries   strain  strain  of Nanopore, obtaining at least 2000 reads with Oxford Nanopore MinION FLO‐MIN106 technology. We obtained the different operational taxonomic units (OTUs) and compared them with the Ribosomal Database Project of Michigan State University. The. syntax file obtained was used to calculate the proportion of each OTU and the reads of the 16S were also analyzed with Epi2me of Oxford Nanopore. Low abundance OTUs  were considered errors/artifacts and removed from the analyses  without any additional source of energy (Fe or S) and 1% e‐scraps w/v. Two flasks were inoculated with the bacterial consortium described previously and two flasks without bacterial inoculum were used as control. Bioleaching trials were maintained in agitation (250 rpm) and at 30°C for 15 days. Bacterial inoculum consisted of 20 ml of a saturated culture of the selected bacterial consortium. During the biolixiviation experiment, the bacterial consortium thrived using e‐scraps as a primary source of energy.Samples were collected on days 7 and 15 since the inoculum. The whole volume of each Erlenmeyer flask was sampled. Samples were filtered with a sterile filter of 0.45 microns of pore size to separate the liquid and solid phases. The liquid phase, leachate, and pellet were analyzed for 45 elements concentration and preserved for the next biouptake experiment. The solid phase was also recovered to analyze the elemental composition and compare it with the starting material.Li) of each element i was obtained as in the following equation , Cie the concentration of element i in the leachate at time e (mg/L), M the mass of the e‐scraps powder sample (g), and V the final volume of the leaching solution (L).Leaching ability (Ei) for each element was thus calculated by referring the leaching ability to the initial concentration in the e‐scraps powder, as given in the following equation  or fresh BG‐11 media in the controls. Starting pH values of the study and control dissolutions were 3.55. The initial inoculum consisted of ~3.5 ± 1.1 × 10The whole volume of one vial of each one of the three groups  was sampled at four different times: 0, 6, 144, and 288 h respectively. Each sample was processed in the following manner: first, an aliquot of the vial was used to check the appearance of the cultures and verify the viability of the cells with optical microscopy . Cellular densities were directly counted using a hemocytometer under the microscope  at 220°C that was performed in microwaves of high pressure. Analyses were performed with an Inductively Coupled Plasma Optical Emission Spectrometry . Liquid samples were dissolved in HNO3 before analyses. Metal concentrations of liquid samples were performed using an Inductively Coupled Plasma Mass Spectrometry . Cellular pellets were digested in an acidic medium (0.2 ml of HNO3 65% Suprapur) for 24 h at 40°C, in Teflon pumps Savillex. Later the analyses were performed with ICP‐MS.Multiple metal screening was conducted in the two‐phase process‐ bioleaching and bio‐uptake‐ to extract metals from e‐scraps. Metal analysis of solid samples required digestion in aqua regia solution —glutaraldehyde (2.5%) for an hour at 4°C (pH was not adjusted and kept at pH ~3), postfixed samples were washed with H33.1E‐scraps were chemically characterized to identify the metals concentrations, presenting a complex composition. E‐scraps metallic content consisted mainly of zinc (7.3%), aluminum (5.9%), titanium (5.2), magnesium (4.6%), and important amounts of barium (3.35%), manganese (3.3%), lead (1.6%), and copper (1%), among others . For the present study, we considered the role of the rare bacteria in the bioleaching process as negligible. The two major species identified in the consortium were A. multivorum (66% of the OTUs) an acidophilic chemoorganotrophic bacterium  another acidophilic bacteria known to use the ferrous iron as electron donor  which increased the surface area and improved the oxidative capacity of bacteria  (%)—amount of the metal solubilized/initial metal concentration in the e‐scraps material—were calculated among the investigated metals after 7 and 15 days of exposure 2(OH)6. These minerals are common precipitates in the conditions of these types of reactions . Bio‐capture occurred in 24 out of 33 analyzed elements present in the control removal dissolution for both strains. From the element scrutiny in the liquors, the removal was very satisfactory for many elements. In general terms, both species retained up to µg of Al, Fe, Zn, Pb, Cu, and Mn, and considerable amounts of other metals, with the better overall performance of ChlSG in terms of ng per g of wet biomass (WB) pellets . Although the amount uptaken per cell is better in EugVP, ChlSG uptake larger amounts per biovolume. Taking this into account, and the higher growth rate of ChlSG, we can infer that ChlSG presented better removal efficiency. Chlamydomonas spp. cellular wall displays a strong affinity for metallic cations , Fe, and Pb in both strains. The fact that EugVP was at its exponential phase while ChlSG reached the stationary phase might explain the possible desorption observed at 12 days in ChlSG. Other factors, like initial metal concentration, elements' competition for the bidding sites  and Chlorella vulgaris (74.6 mg/g) and other four species of microalgae. Kucuker et al. (C. vulgaris addressed Nd (157.21 mg/g) and Wojciech Heilmann et al. . The present study is addressing biouptake from complex leachate liquor with living microalgae, solving the pH adaptation mentioned as a bottleneck needed before biosorption , data curation , formal analysis , investigation , writing–original draft (supporting), writing–review and editing . Paloma Martínez‐Alesón: conceptualization (supporting), formal analysis (supporting), investigation , writing–original draft (supporting), writing–review and editing . Victoria López‐Rodas: funding acquisition (lead), supervision , writing–review and editing . Eduardo Costas: funding acquisition (lead), supervision , writing–review and editing . Beatriz Baselga‐Cervera: conceptualization (supporting), data curation , formal analysis , funding acquisition (supporting), visualization (lead), writing–original draft (lead), writing–review and editing (lead).


Significance of total ischemic time (TIT) in the context of ST-segment elevation myocardial infarction (STEMI) is still controversial. Therefore, in this study, we have evaluate the association of TIT with immediate outcomes in STEMI patients in whom recommended door to balloon (DTB) time of less than 90 min was achieved.p = 0.022), number of diseased vessels (p = 0.002), use of intra-aortic balloon pump (p = 0.003), and in-hospital mortality (p = 0.002). Mortality rate was 4.5%, 5.7%, and 7.8% for the patients with TIT of ≤ 120 min, 121 to 240 min, and > 240 min, respectively. Thirty days’ risk of mortality on TIMI score was 4.97 ± 7.09%, 5.01 ± 6.99%, and 7.12 ± 8.64% for the patients with TIT of ≤ 120 min, 121 to 240 min, and > 240 min, respectively.A total of 5730 patients were included in this study, out of which 80.9% were male and median age was 55 [61–48] years. The median DTB was observed to be 60 [75–45] min and onset of chest pain to emergency room (ER) arrival time was 180 [300–120] min. Prolonged TIT was associated with poor pre-procedure thrombolysis in myocardial infarction (TIMI) flow grade (Prolonged total ischemic was associated with higher in-hospital mortality. Therefore, TIT can also be considered in the matrix of focus, along with DTB time and other clinical determinants to improve the survival from STEMI. Both American College of Cardiology (ACC)/American Heart Association (AHA)/Society for Cardiovascular Angiography and Interventions (SCAI) and European Society of Cardiology (ESC) guidelines recommend primary percutaneous coronary intervention (PCI) for early reperfusion of the ST-segment elevation myocardial infarction (STEMI) for patients presenting to emergency room (ER) within 12 h of onset of chest pain . PercutaHowever, less attention was given to the other delays in the timeline of STEMI such as time between onset of chest and PCI capable hospital arrival . The totConsequently, it is well established that DTB time is very important in identifying in-hospital outcomes and long term morbidity and mortality in the STEMI; however, significance of total ischemic time in STEMI patients is still debatable. Moreover, there is paucity of local data which further enlightened its significance for further research. Therefore, the aim of this study was to evaluate the association of TIT with immediate outcomes in STEMI patients in whom recommended DTB time of less than 90 min was achieved.This study was conducted on selected patients from a prospectively collected institutional data registry. A single center hospital-based registry was established in January 2015 at the National Institute of Cardiovascular Diseases (NICVD), Karachi, Pakistan. Prior to inclusion, informed consent was taken explaining purpose and benefits of the study. Collected data were accessible to authorized personnel only and confidentiality was maintained at all stage of extraction of data and results. NICVD is one of the largest public sector tertiary care cardiac hospital and training center in Pakistan where primary PCI is being offered as the standard treatment 24/7 since 2010. All the procedures were performed as per the ACC/AHA guidelines by consultant cardiologist with experience of more than 5 years. Any contraindication to dual antiplatelet therapy was ruled out by taking brief history of the patient and an echocardiographic screening was done to assess any potential mechanical complications.Study was commenced after approval of the institutional ethical review committee. In this study, we included patients presented within 12 h of onset of chest pain, diagnosed with STEMI and underwent primary PCI during 1st June 2015 to 28th February 2018. Patients with prior history of any cardiac related surgery or intervention and patients with DTB time of > 90 min, due to any reason, were also excluded from the study. The TIT was measured on hospital arrival and was defined as the duration (in minutes) between onset of chest pain and ER arrival plus the duration (in minutes) between ER arrival and device activation. The DTB time was defined as the duration (in minutes) between ER arrival and device activation. Variables extracted from the registry were patient’s demography, risk profile, intra procedural characteristics, post procedural components, presenting KILLIP class, time variables, and in-hospital mortality.p ≤ 0.05 was the criteria for statistical significance.Patients were grouped into three based on TIT: Group 1, TIT ≤ 120 min; Group 2, TIT between 121 to 240 min; and Group 3, TIT > 240 min. All the variables were entered into the IBM SPSS Statistics for Windows, Version 21.0.  for data analysis. Frequency and percentages were calculated for all the categorical variables. Continuous variables were assessed for normality of distribution by applying Kolmogorov–Smirnov normality test and appropriate summary measure such as mean ± standard deviation (SD) or median [interquartile range (IQR)] were computed. Records with missing information regarding disease anatomy, in-hospital outcome, and other study variables were excluded from the analysis. The receiver operating characteristic (ROC) curve analysis was performed and area under the curve (AUC), optimal prognostic cutoff value of TIT and its sensitivity and specificity were calculated. Chi-square test was performed to assess the differences in various characteristics and outcomes by different TIT groups. Multivariable logistic regression analysis was performed to assess the predictors of mortality taken demographic characteristics (such as gender and age), co-morbid conditions , and TIT as explanatory variables based on results of univariate comparison and clinical judgment and odds ratio (OR) and 95% confidence interval (CI) were reported. Throughout the analysis, p < 0.001), gender (p < 0.001), diabetes (p = 0.039), and hypertension (p = 0.021). Baseline clinical and demographic characteristics stratified by the total ischemic time are presented in Table A total of 5730 patients were selected, of them 4637 (80.9%) were males and median age was 55 61–48) years. The median of DTB was observed to be 60 (75–45) min and chest pain ER arrival time was 180 (300–120) min. A total of 2978 (52%) patients were hypertensive, 1712 (29.9%) diabetic, 1636 (28.6%) smokers, and 263 (4.6%) had family history of CAD. Prolonged TIT was found to be associated with age (–48 yearsp = 0.022), number of diseased vessels (p = 0.002), use of IABP (p = 0.003), and side branch involvement (p = 0.008).Angiographic and procedural characteristics stratified by the TIT are presented in Table Post-procedure TIMI flow grade 0 was observed in 94 1.6%), I in 47 (0.8%), II in 228 (4.0%), and III in 5361 (93.6%) patients. In-hospital mortality rate was 388 (6.8%) and mortality rate was 4.5%, 5.7%, and 7.8% for the patients with TIT of ≤ 120 min, 121 to 240 min, and > 240 min, respectively. Thirty days risk of mortality on TIMI score was 2.2% (4.4–1.6%), 2.2% [4.4–1.6%], and 4.4% [7.3–2.2%] for the patients with TIT of ≤ 120 min, 121 to 240 min, and > 240 min, respectively. The optimal cutoff value of TIT based on ROC curve analysis was found to be 297 min (rounded of to 300 min), with sensitivity of 50.26% and specificity of 63.33%. ROC curve and in-hospital mortality rate stratified by the TIT are presented in Fig. .6%, I inp = 0.046), age , diabetes , and TIT  were significant predictors of in-hospital mortality.Multivariable logistic regression analysis for in-hospital mortality is presented in Table In this study, our aim was to assess the effect of TIT on mortality of STEMI patients in whom the recommended DTB time of < 90 min was achieved. We found that TIT is a good prognostic marker, mortality rate is linearly associated with TIT and every delay of an hour after onset of chest pain increases risk of mortality nearly by 5% (OR = 1.05). The optimal prognostic cutoff value for the TIT was 300 min, after which the risk of mortality increases exponentially. In our setup, more than half (54.3%) of the STEMI patients had TIT of more than 4 h. Prolonged TIT was observed to be associated with age and female gender, and patients with prolonged TIT had low TIMI flow grades, multivessel involvement, and increased use of IABP.p < 0.05) as compared to alive patients. Results from a Korean nationwide registry by Kim et al. [The prognostic importance of TIT is well documented by various studies in the past. De Luca et al.  reportedm et al.  observedm et al. , utilizaHence, most of the studies addressing the impact of TIT in STEMI management, including our study, agreed to the fact that TIT holds prognostic utility. In fact, in a number of cases, it proved to be a better predictor of mortality  than DTB time , 13–16. Single center registry-based study and non-randomized nature of the design are key limitations of this study. Secondly, time of onset of chest pain was noted in the ER based on patient or attendants recall, which may have some over or understating. Even though TIT was observed to be an independent predictor of in-hospital mortality, but low predictive value of ischemic time warranted further investigation for the potential effect of confounding factors.Prolonged TIT was associated with higher in-hospital mortality of STEMI patients in whom the recommended DTB was achieved. Although predictive value of TIT was low, but it was found to be an independent predictor of mortality and risk of mortality increases nearly by 5% with every passing hour of TIT. This could be achieved through a rigorous Emergency Management System (EMS) and meticulous patient awareness programs in developing countries.


Antiresorptive treatment has been shown to impair mucosal cell proliferation, migration, and viability. However, in the clinic, antiresorptives are often used in combination with other drugs. We studied the effect of antiresorptives combined with a corticosteroid or antiestrogen on oral mucosal keratinocytes and fibroblasts.Human gingival keratinocyte and fibroblast cell lines were exposed to bisphosphonates (BPs) and denosumab in different concentrations and durations together with an antiestrogen or corticosteroid. Changes in cell viability, proliferation and migration after exposures were measured. Data were evaluated with hierarchical linear mixed model for repeated measurements.Bisphosphonate exposure suppressed keratinocyte and fibroblast cell viability, proliferation, and migration in a time‐dependent manner. Combining a corticosteroid or antiestrogen with BPs further increased this negative effect. Denosumab alone had a mild positive effect on keratinocyte and fibroblast growth. When denosumab was combined with a corticosteroid or antiestrogen, cell growth was suppressed.Our results show that coexisting medications may increase the negative impact of BPs or denosumab on oral mucosal cells. BPs have been shown to impair mucosal cell proliferation, migration, and viability in 12 studies  is a potential complication of BP/denosumab therapy and a growing problem  with 1% Penicillin‐Streptomysin‐Neomycin, 1% L‐glutamine, and 10% Fetal Bovine Serum  for fibroblast cells and Keratinocyte‐SFM Medium (Kit) with l‐glutamine, Epidermal Growth Factor and Bovine Pituitary Extract  for HMK cells.Spontaneously immortalized human gingival keratinocytes (HMK cell line)  and Pamidronate , were used in gradient concentrations of 0, 5, 100, and 500 μmol/L. The antiresorptive Denosumab  was used in gradient concentrations of 0, 6, 25, and 600 μg/mL. These concentrations were chosen to cover a wide range of possibilities since no consensus exists on local tissue levels . These time points were determined from previous studies  according to the manufacturer's protocol. The unit of these spectrophotometric tests was absorbance in accordance with the manufacturer's protocol. Both assays were conducted in duplicate with four separate replicates.Cell viability was tested with AlamarBlue®  at a concentration of 10%. Experimental detection of AlamarBlue dye was made through spectrophotometry (wave length of 570 nm) after an incubation time of 3 h. The cell media were changed at every assay time point. After the AlamarBlue detections, the cells were assayed for proliferation (CellTiter 96® AQ2.4Cells (confluence 100%) exposed to BPs  and denosumab  with and without corticosteroid were tested with migration assay Incucyte  according to the manufacturer's protocol. The cells were monitored for 24 h. The migration process was automatically recorded with IncuCyte Zoom Software and relative wound density data for statistical analysis. The results were analyzed at the time points of 8, 16, and 24 h after “wounding” with relative wound density percentages, as recommended by the manufacturer. The assay was conducted in eight separate replicates.2.5All results were recorded in Microsoft Excel , with the exception of the wound healing results, which were analyzed with the appropriate IncuCyte software. Analyses were further performed using SAS System, version 9.4 for Windows .In the migration experiment, changes in absorbance and relative wound density were evaluated with hierarchical linear mixed model for repeated measurements. Drug exposure was handled as a between‐factor and time point of measurement  as a within‐factor. Interaction between drug exposure and time was also included in the model. The same modeling techniques were used to analyze viability and proliferation. Concentration, time, and drug exposure were included in the model. All statistical tests were performed as two‐sided, with the significance level set at 0.05.2.6This article does not contain any studies with human participants or animals performed by any of the authors, and therefore, no ethics approval was needed.2.7This article does not contain any studies with human participants or animals performed by any of the authors, and therefore, no consent was needed.33.1p < .03). Combined exposure to BP and corticosteroid further suppressed viability in keratinocytes, particularly after 24–48 h . In a similar fashion, all BP concentrations combined with antiestrogen resulted in reduced keratinocyte viability relative to BP or antiestrogen exposure alone   . Denosumab combined with antiestrogen also reduced keratinocyte viability 24 h after exposure at all concentrations  compared with denosumab exposure alone (p < .0082) and compared with antiestrogen alone the result was not significant (p > .62).Denosumab exposure alone significantly increased the viability of keratinocytes at nearly all of the concentrations and exposure times investigated. However, when denosumab was combined with corticosteroid, a significant decrease in keratinocyte viability was detected compared with denosumab alone  Figure . Relativp < .0001) (Table p < .0001). Decreased viability was detected in fibroblasts exposed to antiestrogen alone 24 h after exposure, but not at later time points. Combined antiestrogen and higher BP concentrations decreased fibroblast viability  compared with either BP or antiestrogen exposure alone. The impact of combined BP and corticosteroid exposure on fibroblast viability was somewhat inconsistent, but always greater than that of BP or corticosteroid exposure alone .Both BPs radically decreased the viability of fibroblasts in a time‐dependent manner p < .000 (Table 1p < .05). Combined denosumab and corticosteroid exposure decreased fibroblast viability relative to denosumab exposure alone . Compared with corticosteroid alone, no significant differences emerged.Denosumab exposure resulted in inconsistently increased viability in fibroblasts Table  (p < .053.2p < .0001) and especially at higher concentrations (100 and 500 μg/mL) (Tables p < .0006) (Table p < .0082). All BP concentrations combined with antiestrogen further decreased proliferation compared with BPs  or antiestrogen alone   Tables . Cortico6) Table . Keratinp < .0167). Combined denosumab and corticosteroid exposure decreased keratinocyte proliferation relative to denosumab alone (p < .0001), but not relative to corticosteroid alone. Denosumab combined with antiestrogen reduced keratinocyte proliferation at all time points and concentrations in comparison with denosumab exposure alone (p < .0001). Compared with antiestrogen alone, no significant differences emerged (p > .5549).Denosumab exposure at the highest concentration increased the proliferation of keratinocytes (p < .0067). Denosumab exposure at lower concentrations increased fibroblast proliferation. Corticosteroid exposure alone also suppressed fibroblast proliferation. Antiestrogen‐exposed fibroblasts had a lower proliferation than negative controls 24 h after exposure (p < .0001). The effect of combined corticosteroid and BP exposure on fibroblast proliferation was inconsistent, but always negative relative to BP or corticosteroid exposure alone. The effect of BPs with antiestrogen was inconsistent in fibroblast proliferation.BPs markedly decreased fibroblast proliferation in relation to elapsed time and at all concentrations . There was no effect of combined denosumab and corticosteroid exposure on fibroblast proliferation. There was also no effect of combined exposure of denosumab and antiestrogen on fibroblast proliferation .Denosumab exposure inconsistently increased fibroblast proliferation (3.3p < .001) even 72 h after zolendronate (100 μmol/L) exposure. Migration was also decreased with corticosteroid alone. With combined BP and corticosteroid exposure, migration of keratinocytes was further impaired (p < .0018) (Table Keratinocyte migration was decreased after BP exposure. It was 59% lower than in negative controls p < .00 even 72 p < .04).Migration of only denosumab‐exposed keratinocytes did not differ from negative controls. Combined denosumab‐ and corticosteroid‐exposed keratinocytes showed impaired migration after 8 h, but not at other time points (p < .001). Fibroblast migration was further impaired with combined BP and corticosteroid exposure (p < .0001).Migration of fibroblasts was impaired after BP exposure. Similarly, it was decreased after corticosteroid alone  of zolendronic acid is dose‐dependent, ranging from 403 to 2252 ng/mL  of 6 μg/mL occur in 10 days, but in the case of malignancies the dosage is twofold higher and administered two to six times more often , reducing the possibility of significant errors in cell culture.The main strength of this study was that the evaluation of the effects of BP and denosumab on gingival cells was performed using a wide variety of concentrations and measurement time points postexposure and three parallel tests, increasing the reliability of the results. Furthermore, we used gingival cells representing the exact location of MRONJ in the clinical setting. The number of individual cell cultures was high (denosumab The major challenge in designing this study was the determination of reagent concentrations, as noted earlier in the discussion. This is due to the uncontrolled release of BPs in excessive bone resorption, therefore causing unpredictable concentrations affecting the oral mucosa. Measuring effects of drug exposures with analysis systems became challenging at the highest BP concentrations since cell confluence decreased radically. A further limitation of the study was the use of monolayer cell cultures. However, monolayer culture is the first step in revealing the phenomenon behind the clinical situation. In any case, further investigations using in vitro 3D‐modeling are needed to map the interactions between cells in MRONJ.Our results show that corticosteroid and antiestrogen may increase the negative impact of BP or denosumab on oral mucosal cells. This offers one possible explanation for the epidemiological findings that corticosteroid and antiestrogen therapy contribute to the risk of MRONJ.The authors declare no conflict of interest.Authors TS and JR conceived the study concept. JR and HME developed the theory. HME conducted the experiments with laboratory assistance. EL performed the computations and verified the analytical methods, and HME reported the analytical findings. TS and JR supervised the work. HME drafted the manuscript. All authors discussed the results and contributed to the final manuscript.