In newly diagnosed paediatric patients with moderate-to-severe Crohn’s disease (CD), infliximab (IFX) is initiated once exclusive enteral nutrition (EEN), corticosteroid and immunomodulator therapies have failed. We aimed to investigate whether starting first-line IFX (FL-IFX) is more effective to achieve and maintain remission than conventional treatment.In this multicentre open-label randomised controlled trial, untreated patients with a new diagnosis of CD  >40) were assigned to groups that received five infusions of 5 mg/kg IFX at weeks 0, 2, 6, 14 and 22 (FL-IFX), or EEN or oral prednisolone  . The primary outcome was clinical remission on azathioprine, defined as a wPCDAI <12.5 at week 52, without need for treatment escalation, using intention-to-treat analysis.100 patients were included, 50 in the FL-IFX group and 50 in the conventional group. Four patients did not receive treatment as per protocol. At week 10, a higher proportion of patients in the FL-IFX group than in the conventional group achieved clinical  and endoscopic remission . At week 52, the proportion of patients in clinical remission was not significantly different (p=0.421). However, 19/46 (41%) patients in the FL-IFX group were in clinical remission on azathioprine monotherapy without need for treatment escalation vs 7/48 (15%) in the conventional group (p=0.004).FL-IFX was superior to conventional treatment in achieving short-term clinical and endoscopic remission, and had greater likelihood of maintaining clinical remission at week 52 on azathioprine monotherapy.NCT02517684).ClinicalTrials.gov Registry ( Crohn’s disease is an incurable, debilitating IBD that presents during childhood or adolescence in 8% of all patients with Crohn’s disease.In adult patients with Crohn’s disease, early infliximab (IFX) treatment has shown to affect the natural course of the disease, with a decrease in the occurrence of disease complications.Although corticosteroids are known to negatively impact growth and development in children and adolescents, IFX is currently reserved for corticosteroid and immunomodulator refractory paediatric Crohn’s disease.Ten weeks after start of therapy, the proportion of children in clinical and endoscopic remission is significantly higher in the group treated with first-line IFX than in the group that received exclusive enteral nutrition or prednisolone .First-line IFX treatment is superior in achieving clinical remission on azathioprine monotherapy at 1 year without the need for corticosteroids or further biologics. A significant proportion of children in the conventional treatment group received an additional course of corticosteroids.Despite the possibility to escalate to treatment with IFX, the group that received conventional treatment shows poorer growth at 1 year.This study, being the first randomised controlled trial that investigates the effectiveness of first-line IFX in therapy-naïve paediatric patients with Crohn’s disease, argues that children with moderate-to-severe Crohn’s disease would benefit from first-line IFX treatment.Considering the detrimental impact of an insufficiently effective treatment strategy on growth, development and school attendance of these children and adolescents, a maximally effective therapy from diagnosis onwards is highly desired. This study provides evidence for starting IFX therapy in children with newly diagnosed moderate-to-severe Crohn’s disease.In newly diagnosed paediatric patients with Crohn’s disease (CD), rapid disease control is desirable, but this outcome is not always achieved with current treatment strategies. The European Society of Paediatric Gastroenterology, Hepatology and Nutrition consensus guideline recommends starting with exclusive enteral nutrition (EEN) or oral corticosteroids for induction of remission in conjunction with immunomodulator maintenance treatment. Infliximab (IFX), an anti-tumour necrosis factor alpha (TNF-α) agent, is increasingly being used in paediatric patients with CD refractory to steroids and/or immunomodulators and results in high-sustained remission rates.In many paediatric patients with CD, however, particularly in those with moderate-to-severe CD, mucosal healing and sustained clinical remission are not achieved with conventional treatment.We hypothesise that induction of remission with FL-IFX in moderate-to-severe paediatric patients with CD results in higher early clinical and endoscopic remission rates, and superior rate of clinical remission maintenance on azathioprine (AZA) monotherapy compared with conventional treatment. Therefore, we aim to compare the efficacy of FL-IFX treatment with conventional treatment in newly diagnosed patients with moderate-to-severe paediatric CD.We designed an investigator-initiated international open-label RCT in adherence to the Consolidated Standards of Reporting Trials statement. The trial was performed in 12 hospitals in three European countries . The study protocol was published.It was aspired to enrol patients as soon as possible following diagnostic endoscopy. After CD diagnosis had been established and eligibility criteria had been met, written informed consent was obtained from the patient (if ≥12 years) and both parents and/or guardians.Included patients were stratified by centre and equally randomised into two treatment groups with a validated variable block randomisation model, incorporated in the web-based database used for this trial (Castor Electronic Data Capture).The FL-IFX group received five intravenous IFX  infusions of 5 mg/kg induction at weeks 0, 2 and 6, followed by two maintenance infusions every 8 weeks. This was combined with oral AZA as maintenance treatment , which was initiated on the day induction treatment was started . Convent10.1136/gutjnl-2020-322339.supp1Supplementary dataIn both groups, data were collected prior to start of induction therapy, at weeks 6, 10, 14, 22 and 52. At each visit, weighted Paediatric Crohn’s Disease Activity Index (wPCDAI) was determined,In case of non-response or absence of response (response being a decrease in wPCDAI of >17.5 points), the treatment advice for FL-IFX-treated patients was to shorten the IFX dosing interval to 6 weeks and/or to double the dose to 10 mg/kg. In accordance with clinical practice, clinicians could perform reactive therapeutic drug monitoring (TDM) to guide this decision. Initiation of IFX treatment was advised for conventionally treated patients. To guide clinical decision-making for treatment escalation, secondary loss of response was defined either by an increase of the wPCDAI with >17.5 points or by a total wPCDAI score >40 after response had been achieved. If FL-IFX-treated patients were not in clinical remission at week 22, it was recommended to continue the IFX infusions as standard care, instead of stopping after five infusions. Patients requiring such extended IFX therapy were considered treatment failures in intent-to-treat analysis of outcomes following five doses of FL-IFX. If FL-IFX-treated patients had loss of response during AZA monotherapy, it was advised to check AZA metabolite levels to assess optimal treatment. Contingent on optimal AZA metabolite levels, it was advised to restart IFX maintenance therapy every 8 weeks, also meaning treatment failure. Conventionally treated patients with loss of response during AZA monotherapy were advised to step up to IFX therapy after checking AZA metabolites and optimising its dosing in case of suboptimal levels. In addition to these guidelines, in patients without response, loss of response or intolerance to treatment, changes in treatment could be made according to the physician’s discretion.The primary outcome of this study was clinical remission, defined as wPCDAI <12.5 at week 52, without need for treatment escalation. Any additional CD-related therapy or surgery during the 52 weeks was considered treatment escalation.Additional CD-related therapy in the FL-IFX group included (1) any course of corticosteroids, (2) increase of the IFX dose, (3) shortening of the IFX treatment interval, (4) continuation or restart of IFX after the standard five infusions, or (5) start of another biological agent. Additional CD-related therapy in the conventional treatment group included initiation of IFX and any course of corticosteroids that was additional to the standard treatment described in the previous section.Secondary outcomes included time-to-treatment escalation from start of induction and clinical disease activity scores over time. At week 10, clinical remission rate, endoscopic remission rate and faecal calprotectin level were assessed. QOL was evaluated at week 14. At week 52, the following outcomes were assessed: (1) additional corticosteroid use, (2) need for treatment escalation, (3) linear growth, (4) clinical remission rate, (5) endoscopic remission rate, (6) faecal calprotectin level, (7) QOL and (8) rate of adverse events. An adverse event was defined as any undesirable experience occurring to a subject during the study, whether or not it was considered to be related to the investigational product or the experimental treatment.2 test or the Fisher exact test. The Wilcoxon signed rank test was used to compare height-for-age SDS at different time points within one treatment group. SES-CD scores with a missing ileum subscore due to the endoscopist’s failure to intubate the terminal ileum were included in the analysis to evaluate endoscopic remission. The median faecal calprotectin levels and SES-CD scores were subject to right censoring. To correct for this, medians of faecal calprotectin levels and SES-CD scores were calculated using the Kaplan-Meier method, and treatment groups for these outcomes were compared using the log-rank test. The multiple imputation method was used for missing erythrocyte sedimentation rate (ESR) levels (14.8%), missing albumin levels (10.5%) and missing faecal calprotectin levels (10.9%) in order to calculate biochemical remission rate. Twenty complete datasets were created for multiple imputation. For the primary outcome, no imputation was performed as <5% of data were missing. The time-to-treatment escalation outcomes were analysed using the Kaplan-Meier method. A paired analysis was performed for the linear growth. The mean clinical disease activity score over time was calculated with a linear mixed model, including the assigned treatment as a fixed effect and intercept as random effect. Random slopes were tested but not included.Based on published studies reporting effectiveness of FL-IFX treatment and early IFX useAll analyses were performed based on a significance level of 0.05. Calculations were performed using IBM SPSS Statistics V.24.0.Patients were recruited between 7 April 2015 and 19 November 2018. A total of 195 patients were screened for eligibility in this trial. One hundred patients were randomly assigned to FL-IFX (n=50) or conventional treatment (n=50) . One patThe median time between diagnostic endoscopy and start of treatment for all included patients was 8 days (IQR 4–14). Twenty-seven patients (56%) in the conventional treatment group received EEN as primary induction therapy, while 20 patients (42%) received prednisolone .Ten weeks after start of induction therapy, significantly more FL-IFX-treated patients than conventionally treated patients were in clinical remission (59% (24/41) vs 34% (15/44), p=0.021). Fifty-seven patients , with similar baseline characteristics , underweThe mean wPCDAI score at all time points, corrected for repeated measurements, was lower in the FL-IFX group than in the conventional treatment group, although not significantly different  . During 10.1136/gutjnl-2020-322339.supp2Supplementary data10.1136/gutjnl-2020-322339.supp3Supplementary dataTwenty-one patients in the FL-IFX group needed treatment escalation. Twelve (24.5%) continued IFX therapy after the five per-protocol infusions . Twenty-Based on reactive TDM 2/49 patients received dose escalation within the first 22 weeks. None of the seven patients that restarted IFX experienced side effects or needed to stop within the first year of follow-up.As depicted in At week 52, the primary outcome of clinical remission without need for treatment escalation was reached in more FL-IFX-treated patients than in conventionally treated patients. In particular, 19/46 (41%) of the FL-IFX-treated patients were in clinical remission without need for treatment escalation, vs 7/48 (15%) of the conventionally treated patients . This reIn contrast, in the FL-IFX-treated patients median SDS height-for-age significantly improved between baseline and week 52 (median SDS of −0.07 (IQR −0.84 to 0.76) at baseline vs 0.02 (IQR −0.81 to 0.70) at week 52, p=0.045), while it significantly decreased in conventionally treated patients (median SDS of −0.53 (IQR −1.06 to 0.26) at baseline vs −0.66 (IQR −1.13 to 0.11) at week 52, p=0.020) .At week 14 and week 52, the median QOL scores in the FL-IFX group and conventional group were similar and in both groups significantly higher at both time points than at baseline . The medThere was no significant difference between the proportion of patients with an adverse event in the FL-IFX group (44%) versus the conventional treatment group . In total, 94 adverse events occurred, 40 of which were reported in FL-IFX-treated patients and 54 in conventionally treated patients. Fifteen serious adverse events were reported .This is the first RCT to compare the efficacy of IFX directly after diagnosis to conventional treatment with corticosteroids or EEN in newly diagnosed paediatric patients with moderate-to-severe CD. Ten weeks after induction therapy, higher clinical remission rates are found in the FL-IFX group. Of the FL-IFX-treated patients that underwent an endoscopy at week 10, a higher proportion is in endoscopic remission than in the conventional treatment group. Overall, the proportion of patients in clinical and biochemical remission at 1 year did not significantly differ between the two treatment groups. However, the trajectory towards remission is very different between groups. In particular, FL-IFX treatment is superior to conventional treatment in achieving clinical remission without need for treatment escalation 1 year after the start of therapy. Children with moderate-to-severe CD benefit from an effective therapy from diagnosis onwards. In this young population, delay in achieving remission and frequent flare-ups in the first year after diagnosis may slow their pubertal development and affect their school attendance and general well-being.et al.In our cohort, endoscopic remission rates in FL-IFX-treated patients were significantly higher at week 10 than those in conventionally treated patients. The endoscopic remission rate of 59% in the FL-IFX group is superior to previously reported endoscopic remission rates in both paediatric and adult studies,et al demonstrated that retreatment with IFX was effective and well-tolerated in a group of patients that stopped IFX treatment after at least 1 year and 6 additional months of corticosteroid-free remission.Since the design of this study in 2015, guidelines were updated and the role of therapeutic drug monitoring has increased. In our study, reactive TDM was performed based on clinical practice and thus the clinician’s decision. In only a few patients this resulted in treatment optimisation, as only 2 out of 49 patients received interval shortening and/or dose escalation within the first 22 weeks. Thus far, IFX could be safely restarted in the patients in our cohort. However, the risk of increased immunogenicity and consecutive loss of response after restarting IFX has been demonstrated and longer follow-up of our cohort is needed.Counterarguments for implementation of FL-IFX therapy could be the increased risk of side effects and higher costs.A clear strength and innovative aspect of this study is the inclusion of new-onset and therapy-naïve patients with CD. Performing an RCT in children with CD is rarely done and extremely challenging. Only 21 patients and their parents declined to participate in this trial, which demonstrates the patients’ interest in FL-IFX treatment. There are some drawbacks associated with our study. First, treatment assignment and assessments guiding treatment changes were not masked for patients and investigators, which could have created a performance bias. However, this bias is partly mitigated by the evaluation of growth, faecal calprotectin and endoscopic remission as objective outcome measures in this study. Second, while participating in the study, not all patients agreed on the endoscopic evaluation scheduled in this study at weeks 10 and 52. Based on ethical considerations, these patients continued to participate in the study, which led to missing endoscopic results. Despite the lower numbers, this did not introduce a bias as patients who underwent endoscopy had comparable disease characteristics at baseline . Third, In conclusion, despite the similar clinical remission rates at 1 year after diagnosis in both treatment groups, we argue that children and adolescents with moderate-to-severe CD would benefit from FL-IFX treatment as an insufficiently effective treatment strategy impacts their growth and development. Ongoing disease activity or corticosteroids use prior to escalation to IFX in the conventional treatment group could have been prevented by starting FL-IFX. This innovative treatment was well accepted by children and their parents, which shows the importance of moving forward with protocols to allow us to learn what is best. Future follow-up and additional research are needed to determine whether IFX can be stopped and for which patients this will be beneficial.


Background: Proprotein convertase subtilisin/kexin type 9 inhibitor alirocumab reduce ischemic events; however, the cost-effectiveness remains uncertain. This study sought to evaluate its economic value in patients with myocardial infarction (MI) from the Chinese healthcare perspective.Methods: A state-transition Markov model was developed to determine the cost-effectiveness of alirocumab for preventing recurrent MI, ischemic stroke and death. Preventative effect of the therapy was gathered from ODYSSEY OUTCOMES trial and absolute reduction of low-density lipoprotein cholesterol (LDL-C) in ODYSSEY EAST trial, respectively. The primary outcome was the incremental cost-effectiveness ratio (ICER), defined as incremental cost per quality-adjusted life-year (QALY) gained.Results: Compared with statin monotherapy, the ICER of alirocumab therapy at its present discounted price  based on clinical follow-up efficacy was 1,613,997 CNY per QALY gained. A willingness-to-pay threshold of 212,676 CNY per QALY would be achieved when the annual cost of alirocumab was reduced by 88% from the full official price to 6071 CNY. The therapeutic effect evaluation estimated by the magnitude of LDL-C reduction was superior to the results of clinical follow-up, but this medication was still far from cost-effective. Multiple vulnerable subgroup analyses demonstrated that the ICER for patients with polyvascular disease in 3 vascular beds was 111,750 CNY per QALY gained.Conclusion: Alirocumab is not cost-effective in general MI population based on current discounted price. High long-term costs of alirocumab may be offset by health benefit in patients with polyvascular disease (3 beds). Cardiovascular disease (CVD) plays an increasing role in years of life lost currently, accounting for 40% of deaths in the Chinese population . Low-denIn daily clinical practice, approximately 82.9% of MI patients, from a nationwide Swedish survey, would be eligible for intensive lipid-lowering therapy as not attaining the updated target of LDL-C level  which paAccurate economic evaluation of new therapy is available and necessary to make certain the treatment effect and potential tradeoffs among therapies after the results of the related large randomized controlled trials are released together with the price determined in healthcare system. Although the economic value of alirocumab is inconsistent in the published papers from American groups, substantial price reduction would definitely improve cost-effectiveness regardless of simulated methods . In consWe established a state-transition Markov model to assess the economic value of alirocumab within established MI population . After aAlirocumab plus statin therapy  vs. statin monotherapy were projected through accrued simulation. The incremental cost-effectiveness ratio (ICER), a primary outcome, was calculated by incremental cost per quality-adjusted life-years (QALYs) gained. We assumed the cost-effectiveness threshold as 212,676 Chinese Yuan , which A representative real-world cohort with established MI was modeled to reflect the Chinese high-risk CVD population. Patients were selected from the China PEACE Prospective AMI study  , the firThe effect of alirocumab has been much-discussed in previous research literatures. We aimed to use two approaches to incorporate the efficacy of alirocumab in this health economic model . One wasWe assumed that the initial conversion probabilities above were applied to the first year and increased in every succedent 5 years in line with the growth rates of real-time natural mortalities in all age groups in China Health Statistics Yearbook . All 25-The setting of this economic evaluation was based on Chinese healthcare sector. The direct costs associated with MACEs and subsequent chronic treatment, including initial event-free state, were obtained from previous literatures  and adjuCurrently, public and private medical insurances do not cover PCSK9 inhibitors. Following the usual therapeutic dose and frequency (75 mg/2 weeks), the annual official price of alirocumab was estimated to be 51,532 CNY per person. As the PCSK9 inhibitor manufacturers had offered an average rebate of 33% since April 2020, main base case analysis was performed for discounted net price of alirocumab in 34,355 CNY annually. We also calculated the average bidding price of ezetimibe (10 mg), equally 2827 CNY per year, for scenario analyses.Quality-of-life (QOL) estimates for health states were derived from a time tradeoff study . The disIn one-way sensitivity analyses, we varied single input through plausible range  , while hBased on the ODYSSEY OUTCOMES trial, we made different hypothesis about effect of alirocumab. The efficacy of alirocumab on all-cause death  was applied instead of the efficacy of alirocumab just on cardiovascular death in the base case. We modeled ezetimibe vs. alirocumab based on statin therapy. The efficacy of ezetimibe was modeled according to the IMPROVE-IT trial  and the A range of scenario analyses were performed in high-risk subgroups to identify preferred candidates for the PCSK9 inhibitor. Women in China had higher all-cause/cardiovascular mortalities vs. men at 1-year post-MI , so we eWe conducted all these analyses in disparate prices of alirocumab and simulation ways, if feasible, and key parameters are showed in The model was performed by using TreeAge Pro 2020 R1.1 software program . We followed the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) guidelines  and the In the main result applying the HRs observed in ODYSSEY OUTCOMES to the model, mean costs would be 871,321 CNY with alirocumab added to statin therapy and 400,705 CNY with mere statin therapy. The clinical benefit of alirocumab were 0.29 gained QALYs. Compared with statin monotherapy, alirocumab at its present price  was estimated to produce an ICER of 1,613,997 CNY per QALY gained . If, insIn one-way sensitivity analyses, the main drivers on the ICER of alirocumab therapy at its present price based on clinical follow-up efficacy were the utility of initial event-free and post-IS . AdditioProbabilistic sensitivity analyses were demonstrated in Monte Carlo simulation scatters plots  and costSeparate scenarios were undertaken to evaluate the treatment benefit . An all-Similar or slightly favorable economic value of the PCSK9 inhibitor was observed in several subgroups, including female, DM or hypertension with MI. For poorly controlled FH patients after MI, the ICER was 254,945 CNY per QALY gained and the price was expected to be 30,299 CNY, which was extremely close to the present acquisition price. In patients with polyvascular disease in 3 vascular beds, the ICER for alirocumab plus statin vs. statin alone was 111,750 CNY, which achieved the accepted WTP threshold. Meanwhile, the valued-based price was 50,734 CNY per year, just a small reduction from the full list price. Regarding to different initial treatment ages, the ICERs based on clinical follow-up efficacy declined from 1,412,372 CNY in 65 years old to 842,649 CNY in 80 years old. Long-term use of alirocumab (over 20 years) brought about relatively better economic benefit in general MI population. The diverse discount rates of 3.5% and 6% produced an annual expected price of 6325 CNY and 5900 CNY, respectively.This is the first decision analysis to comprehensively assess the cost-effectiveness of alirocumab in China for our best knowledge. Despite the prominent cardiovascular benefit of alirocumab observed in the ODYSSEY OUTCOMES trial, its use was inefficient in Chinese healthcare system at present. The therapeutic effect evaluation estimated by the magnitude of LDL-C reduction was superior to the results of clinical follow-up, but this medication was still not cost-effective.Two long-term economic assessments of alirocumab in US have been specially conducted. Kazi et al.  simulateA noteworthy observation in the ODYSSEY OUTCOMES trial is the nominal reduction in all-cause mortality stratified from numerical reduction in cardiovascular and non-cardiovascular death. We assumed that the reduction of all-cause death was mediated through the reduction in the risk of cardiovascular death in main outcomes, which has an explicit mechanism induced by lip-lowering consisted with the CTTC meta-analysis . HoweverThe economic value of evolocumab, another previously approved PCSK9 inhibitor in China, has been extensively studied and discussed in fully comparable methods . DespiteNew expensive treatments which cure diseases or improve disability symptoms may be more compatible with their high prices. Delaying the process of atherosclerosis neither cure CVD nor reduce disability, so the potential benefits of PCSK9 inhibitors are limited. Less than one third of the patients in US complied with their prescriptions because of low approval rates or excessive out-of-pocket costs . There iThis study had several limitations. Our cohort selected from PEACE study had only one-year follow-up duration, which may overestimate the incidence of adverse events after MI and the ICERs would be even higher. In addition, the PEACE researchers did not describe the initial average LDL-C level of their cohort. To overcome the shortcomings, we used the absolute LDL-C reduction from the ODYSSEY EAST study involving Chinese population to simulate the effect of alirocumab. And we did not perform subgroup analyses in patients with baseline LDL-C level over or below 100 mg/dL for the above reason, just as a significant difference of therapeutic effect reported in ODYSSEY OUTCOMES trial. These limitations could not affect the conclusion that alirocumab was also not cost-effective in patients with initial LDL-C > 100 mg/dL accounted for the lack of economic value in MI patients with FH defined as higher LDL-C level over 200 mg/dL. In real-world clinical practice, the adherence of patients may decline based on over optimism of the condition or exorbitant costs. From an economic standpoint, there is no increase in cost or benefit when patients stop taking the medicine and therefore do not change the economic outcomes. If the legacy effect of PCSK9 inhibitors is proved, cost-effectiveness will likely rise. Lastly, our analysis is not generalizable to primary prevention or patients with stable CVD.At its current discounted price of 34,355 CNY annually, the addition of alirocumab to statin therapy in MI patients do not attain the generally accepted cost-effectiveness threshold. If pragmatic strategies can be carried out to control cost of alirocumab to 6071 CNY  or 11,861 CNY (assessed from LDL-C reduction), this agent would be a promising treatment option for high-risk patients. Expensive lifetime drug costs may be offset by clinical benefit in patients with polyvascular disease in 3 vascular beds.


There have been concerns regarding the safety of renin-angiotensin-aldosterone-system (RAAS)-blocking agents including angiotensin-converting enzyme inhibitors (ACEI) and angiotensin receptor blockers (ARB) during the coronavirus disease 2019 (COVID-19) pandemic. This study sought to evaluate the impact of hypertension and the use of ACEI/ARB on clinical severity in patients with COVID-19.A total of 3,788 patients aged 30 years or older who were confirmed with COVID-19 with real time reverse transcription polymerase chain reaction were identified from a claims-based cohort in Korea. The primary study outcome was severe clinical events, a composite of intensive care unit admission, need for ventilator care, and death.Patients with hypertension  were older and had higher prevalence of comorbidities than those without hypertension. The risk of the primary study outcome was significantly higher in the hypertension group, even after multivariable adjustment . Among 1,044 patients with hypertensive medical treatment, 782 (74.9 %) were on ACEI or ARB. The ACEI/ARB subgroup had a lower risk of severe clinical outcomes compared to the no ACEI/ARB group, but this did not remain significant after multivariable adjustment .Patients with hypertension had worse COVID-19 outcomes than those without hypertension, while the use of RAAS-blocking agents was not associated with increased risk of any adverse study outcomes. The use of ACE inhibitors or ARBs did not increase the risk of adverse COVID-19 outcomes, supporting current guidance to continue these medications when indicated. The coronavirus disease 2019 (COVID-19) is a global pandemic causing millions of deaths worldwide as of March 2021 . StudiesAmid the COVID-19 pandemic, concerns have been raised that the use of renin-angiotensin-aldosterone-system (RAAS)-blocking agents, such as ACE inhibitors (ACEI) and angiotensin receptor blockers (ARB) may increase the susceptibility to or aggravate the severity of COVID-19. There have been studies suggesting blockade of either angiotensin II synthesis or activity increases the gene expression and activity of ACE2 , 7. ObseMajor cardiology societies pointed out that such speculations lack sound clinical evidence, and strongly recommended that physicians and patients should continue treatment with their usual antihypertensive therapy , 9. HypeSouth Korea is one of the countries severely affected by COVID-19: more than 20,000 patients have been diagnosed and approximately 380 died as of September 2020. In response to scientific needs, the government of Korea allowed access to de-identified COVID-19 nationwide patient data based on the Korean National Health Insurance System . The insA subject was considered to have hypertension if (a) hypertension was diagnosed once or more during a hospitalization, or at two or more outpatient clinic visits; or (b) a history of antihypertensive medication prescription for more than 1 month during the prior 1 year. Subjects with antihypertensive medical treatment were classified into an ACEI/ARB or non-ACEI/ARB group according to the prescription in the year prior: ACEI/ARB use was necessary regardless of combined use of other antihypertension medications to suffice the ACEI/ARB group, while non-ACEI/ARB group definition included any antihypertension drugs other than ACEI or ARB. Previous medical history, such as ischemic heart disease, stroke, congestive heart failure, chronic obstructive pulmonary disease, and cancer, was identified based on International Classification of Diseases-10 codes from the previous 5-year claim data . The priP < 0.05 was considered statistically significant. Statistical analyses were performed using SAS ver. 9.2 .Summary statistics are reported as means ± standard deviations for numerical variables and as numbers (percentages) for categorical variables. Continuous variables were compared using the Student t-test or the Mann-Whitney U-test, as appropriate. Categorical variables were compared using the chi-squared test. Logistic regression models were used to calculate odds ratios (ORs) and 95 % confidence intervals (CIs). Multivariable regression models were constructed with adjustment for (a) age (continuous) and sex, and (b) including age, sex, diabetes mellitus, chronic kidney disease, chronic obstructive pulmonary disease, and cardiovascular disease. A two-sided Among 3,788 adults aged 30 years or older who were diagnosed with COVID-19, 1,190 (31.4 %) had hypertension  were prescribed ACEI or ARB Fig. ; Fig. 2.Table The present study showed patients with hypertension more frequently experienced adverse outcomes than those without hypertension when they have COVID-19 illness. The excess was greater with severe events such as ICU admission, ventilator care, and death, whereas it was less prominent with less severe events such as hospitalization and oxygen requirement. Another important finding of this study is that the use of RAAS-blocking agents such as ACEI and ARB was not associated with increased risk of poor clinical prognosis associated with COVID-19 illness.The present study showed that use of RAAS-blocking agents does not adversely affect clinical outcomes during COVID-19 illness. Several recent studies consistently showed reassuring results regarding the safety of ACEI/ARB during the pandemic. Two studies from China also analyzed hospitalized patients and found no excess in mortality associated with the use of ACEI/ARB , 19. SevThe current evidence of ACE2 upregulation after chronic exposure to ACEI and ARB is mostly focused on cardiovascular tissue , 7. TherPrevious studies have shown that hypertension is the most frequent comorbidity among patients with COVID-19 and that it is also frequent in those with adverse clinical outcomes , 21. ThiThis study similarly showed hypertension was the most frequent comorbidity in this study population. However, the proportion (31.4 %) was numerically similar to the prevalence in the general Korean population (30.5 % in adults aged 30 years or older) , 22. RegOne of the strengths of the present study is the broad spectrum of study population including outpatients as well as in-hospital patients. Vigorous contact-tracing and extensive testing were performed in Korea . The lowThis study showed hypertension is associated with poor clinical outcomes in patients with COVID-19. However, the use of RAAS-blocking agents, including ACEI and ARB, was not associated with increased risk of any adverse study outcomes. The present study supports the current guidance to continue ACEI or ARB during the COVID-19 pandemic.


Arabidopsis gene EMBRYO DEFECTIVE 2726 (EMB2726). Heterozygous emb2726 seedlings produced about 25% albino seeds with embryos that displayed defects at the 32-cell stage and that arrested development at the late globular stage. EMB2726 protein was localized in chloroplasts and was expressed at all stages of development, such as embryogenesis. Moreover, the two translation elongation factor Ts domains within the protein were critical for its function. Transmission electron microscopy revealed that the cells in emb2726 embryos contained undifferentiated proplastids and that the expression of plastid genome-encoded photosynthesis-related genes was dramatically reduced. Expression studies of DR5:GFP, pDRN:DRN-GFP, and pPIN1:PIN1-GFP reporter lines indicated normal auxin biosynthesis but altered polar auxin transport. The expression of pSHR:SHR-GFP and pSCR:SCR-GFP confirmed that procambium and ground tissue precursors were lacking in emb2726 embryos. The results suggest that EMB2726 plays a critical role during Arabidopsis embryogenesis by affecting chloroplast development, possibly by affecting the translation process in plastids.Embryogenesis is a critical developmental process that establishes the body organization of higher plants. During this process, the biogenesis of chloroplasts from proplastids is essential. A failure in chloroplast development during embryogenesis can cause morphologically abnormal embryos or embryonic lethality. In this study, we isolated a T-DNA insertion mutant of the  Arabidopsis, embryogenesis starts from the asymmetric division of a zygote to generate a large basal cell and a small apical cell  was used as the wild-type (WT) control in this study. Seedlings were grown in a growth room under 16 h of light/8 h of darkness (90 μmol m–2 s–1 intensity) at 22°C and 55% relative humidity.emb2726-5/+ and emb2726-6/+ mutations, an egg cell-specific promoter-controlled CRISPR/Cas9 system was used according to To generate the pEMB2726:EMB2726-GUS and pEMB2726:EMB2726-YFP, the EMB2726 genomic sequence  was amplified by PCR using the primers listed in TM8/GW/TOPOTM  following the instructions of the manufacturer. Then, the cloned sequence was subcloned into the binary vectors, pMDC163 .To produce the native promoter-driven constructs  pMDC163  and pEar pMDC163 , using GAgrobacterium tumefaciens strain GV3101 and transformed into Col-0or emb2726-4/+ plants using the floral dip method.The abovementioned expression constructs were introduced into the 2HPO4, and 0.24 g/L KH2PO4, pH 7.5). They were then vacuum-infiltrated for 15 min and kept at room temperature overnight. The seeds were then removed from the fixed siliques under a stereomicroscope and cleared using a chloral hydrate:glycerol:water solution  based on the study of For embryo observation, siliques at 2–6 days after pollination (DAP) were sliced open and immersed in 4% glutaraldehyde in phosphate-buffered saline , green fluorescent protein , and chlorophyll autofluorescence .pEMB2726:EMB2726-GUS transgenic seedlings were vacuum-infiltrated for 30 min in a GUS staining solution  and stained for 1 h (7-day-old seedlings and rosette leaves) or 5 h  at 37°C. The stained tissues were cleared with 70% ethanol and photographed.Tissues from homozygous emb2726-4/+ siliques at 7 DAP using an Eastep® Super Total RNA Extraction Kit  following the instructions of the manufacturer. Complementary DNA (cDNA) was synthesized from 0.5 to 1 μg of total RNA using the Revert Aid First Strand cDNA Synthesis Kit  with oligo (dT)16 primer. Quantitative PCR was performed using SYBR Premix Ex Taq . The sequences of the gene-specific primers used are listed in PP2AA3 was used as an internal control.Total RNA was isolated from green or white seeds in emb2726-4/+ siliques at 5 DAP were fixed, embedded, and sectioned according to the method described by Green and white seeds from Ultra-thin sections were prepared using Leica Ultracut R Ultramicrotome  and collected on formvar-coated nickel grids. The sections were post-stained with saturated uranyl acetate for 15 min followed by lead citrate for 15 min before being examined and imaged with a Hitachi H-7650  transmission electron microscope.n > 4,000). Subsequent analysis showed that this phenotype came from an additional insertion within the mutant that was tightly linked with the original insertion. The Thermal Asymmetric InterLaced PCR technique was applied to identify the second insertion site in the segregated line. The T-DNA left border was recovered at 348 base pairs downstream of the ATG in At4g29060; the right border was positioned 1,054 base pairs upstream of the start codon for At4g29060, within the first exon of At4g29050. Thus, this T-DNA insertion caused a 1.4-kb deletion between the two genes  and the CRISPR/Cas9 mutant (named emb2726-5) at 10 DAP showed that both mutants contained about 25% albino seeds , compared to 0.07% in WT  from siliques at 14 DAP were highly wrinkled and dark brown in color, indicating that the embryos in the albino seeds had degenerated. Reciprocal crosses of emb2726-4/+ with WT plants revealed no significant defects in the transmission efficiencies of the female and male gametophytes, confirming that the lack of homozygous offspring from this mutant is due to a defect in embryogenesis  and transformed into the emb2726-4/+ background. T1 emb2726-4/+ transgenic plants with high-level protein expression produced siliques with almost no albino seeds, and the number of aborted seeds in the siliques was reduced to nearly the level in WT , while all of the embryos in the albino seeds (n > 300) were still in the globular stage without differentiation  using the CRISPR/Cas9 technique  of albino seeds, and the embryos in these seeds were all arrested at the globular stage as in the other two null mutants  and an EF-Ts protein . PSRP-7,echnique . Similar mutants –E. Theseemb2726 seeds failed to turn green, we suspected a defect in chloroplast biogenesis. By using transmission electron microscopy, we observed the chloroplast structure in WT and mutant embryos isolated from siliques at 5 DAP. In WT embryos, the chloroplasts had an organized thylakoid membrane stacked into grana . This result is consistent with the lack of cotyledon initiation in the mutant embryos.DORNROSCHEN (DRN) is involved in auxin-regulated, embryonic cotyledon development . Consistuishable , 16-cellemb2726-4/+ plants. Of more than 508- to 16-cell embryos observed from emb2726-4/+ siliques, only one embryo did not have any GFP fluorescence. This ratio is dramatically lower than the 25% homozygous mutants in emb2726-4/+, indicating that PIN1-GFP was expressed in emb2726-4 embryos earlier than the 16-cell stage. Beginning at the 32-cell stage, no PIN1-GFP fluorescence could be observed in the mutant embryos (emb2726-4), while WT embryos from the same siliques displayed the correct distribution pattern, as reported previously . In comparison, SCR-GFP fluorescence was not observed in emb2726-4 embryos starting from the early globular stage when using the same excitation power as for WT embryos  is localized in mitochondria, and two (At2g25800 and EMB2726) are localized in chloroplasts. However, an analysis of the At2g25800 protein sequence revealed neither an EF-Ts motif nor homology with known EF-Ts, indicating misannotation of the protein and leaving EMB2726 as the only possible EF-Ts in chloroplasts. It has been shown that the homolog of EMB2726 in C. reinhardtii, PETs protein, can be post-translationally processed into PSRP-7 and an EF-Ts protein and that the latter functions as a guanine nucleotide exchange factor for EF-Tu  whose misexpression is responsible for the observed embryonic lethal phenotype is unclear. The plastid-encoded protein accD, a subunit of the heteromeric enzyme acetyl-CoA carboxylase in plastids and which is required for membrane lipid biosynthesis, is a leading candidate  and columella precursors but not for initial hypophyseal division (emb2726 embryos, the lens-shaped QC precursor cell did not differentiate further and could not be maintained at later stages; thus, the root cap did not form. This phenotype is consistent with the low-level expression of SCR-GFP in emb2726 embryos. Therefore, another possible reason for the failure of root initiation in emb2726 could be the misexpression of SCR in hypophyseal daughter cells.During embryonic root development, initiation of the root apical meristem starts with the asymmetric division of the hypophysis. In division . In emb2emb2726 embryos might be exclusively due to the disturbed distribution of auxin.Nonetheless, the SHR-SCR transcription network is not required for ground tissue initiation; instead, auxin could guide the specification of the first ground tissue cells through MONOPTEROS/ARF5 in an SHR-independent manner . Furtheremb2726 embryos, a disrupted auxin distribution was observed after the 16-cell stage, and disrupted cell patterning was distinctly visible at the 32-cell stage. These findings support the notion that the arrested embryos observed in emb2726 were mainly caused by the disrupted distribution of auxin. Still, factors unrelated to auxin might also be involved in the patterning of emb2726 embryos. For instance, the involvement of auxin in protodermal cell fate has not been reported, but the periclinal division was observed in the protoderm layer as emb2726 embryos grew larger (Overall, in w larger . HoweverArabidopsis embryo development. Disruption of the function of EMB2726 could lead to a loss of EF-Ts activity during plastid translation, leading to failed embryogenesis.The results show that EMB2726 is crucial for the globular to the heart stage transition during The original contributions presented in the study are included in the article/CL performed most of the experiments and analyzed the data. J-XS and CQ participated in the phenotype analysis. BZ, JW, and SW participated in the analysis of the reporter lines. YS designed the experiments and wrote the manuscript. All authors made substantial, direct and intellectual contributions in the study, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: The relative benefit of immediate complete revascularization, staged complete revascularization, and culprit-only percutaneous coronary intervention (PCI) remains unclear in hemodynamically stable patients with ST-segment elevation myocardial infarction (STEMI) and multivessel disease. The aim of this study was to compare the clinical outcomes of the 3 PCI strategies in this population.Methods: We followed a pre-specified protocol (PROSPERO number: CRD42020183801). A comprehensive search of the electronic databases including PubMed, EMBASE and Cochrane Library from inception through February 21, 2020 was conducted. Randomized trials evaluating the comparative efficacy and safety of at least 2 of the 3 PCI strategies were identified. The primary endpoint was the composite of cardiovascular mortality or myocardial infarction (MI) during the longest follow-up. Pairwise and network meta-analyses were performed with random-effects model.Results: Eleven trials including 6,942 patients were analyzed. Pairwise meta-analysis noted that immediate complete revascularization and staged complete revascularization were respectively associated with a 52 and 27% reduction in the risk of cardiovascular death or MI , compared with culprit-only PCI. The risk of cardiovascular death or MI was not statistically different in staged and immediate complete revascularization groups . Network meta-analysis obtained almost similar results compared with pairwise meta-analysis, and immediate complete revascularization had a 77% probability of being the best strategy for reducing cardiovascular death or MI among the 3 PCI strategies.Conclusion: The current evidence suggests that both immediate and staged complete revascularization were associated with a reduction of cardiovascular death or MI compared with culprit-only PCI. Further trials are warranted to directly compare immediate vs. staged complete revascularization in this population.Systematic Review Registration:https://www.crd.york.ac.uk/prospero/, PROSPERO [CRD42020183801]. In patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI), approximately 50% have one or more significant non-culprit vessels –3. BasedNevertheless, the relative benefit of immediate complete revascularization, staged complete revascularization, and culprit-only PCI with regard to the hard endpoints in patients with STEMI and multivessel disease remains an unresolved issue. Although current guidelines give an equal level of recommendation to the two types of complete revascularization strategy , 3, non-We followed a pre-specified protocol (PROSPERO number: CRD42020183801). A comprehensive search of the electronic databases including PubMed, EMBASE and Cochrane Library from inception through February 21, 2020 was conducted by two independent investigators to identify pertinent articles published in English (SY and SW). Conference proceedings for the scientific sessions of the ACC, AHA, ESC, Transcatheter Cardiovascular Therapeutics and EuroPCR were also searched. The following medical subject headings and search terms were used: “myocardial infarction”, “multivessel”, “non-culprit”, “non-infarct”, “percutaneous coronary intervention”, “angioplasty”, “revascularization”, “random”, “randomly” and “randomized”. In addition, we examined the references of the identified articles, relevant reviews and meta-analyses to include other potentially eligible studies.Studies satisfying the following criteria were eligible: (1) hemodynamically stable patients with STEMI and multivessel disease; (2) RCTs evaluating the relative efficacy and safety of at least 2 of the 3 PCI strategies, i.e., immediate complete revascularization (PCI of culprit vessels as well as ≥1 non-culprit vessels during the index procedure), staged complete revascularization , and culprit-only PCI ; (3) trials with follow-up period of > 6 months; and (4) trials reported endpoint data of interest. Studies that allowed either an immediate or a staged complete revascularization in 1 randomization arm were categorized according to the predominant strategy (>75%). Of note, if neither of the two treatment strategies account for > 75% of the patients in a group, the study will be excluded. We also excluded studies only enrolling patients with chronic total occlusion (CTO) in non-culprit vessels or cardiogenic shock.The following data was independently extracted by two authors through a standardized form for each study: first author, year of publication, study design, patient characteristics, quality indicators and clinical outcomes (LF and CZ). Differences in assessments were resolved by discussing with a third investigator (DY). The quality of RCTs were assessed by evaluating the following methodological criteria recommended by the Cochrane Collaboration . Trials The primary efficacy endpoint was the composite of cardiovascular mortality or MI during the longest follow-up. All-cause mortality, cardiovascular mortality, MI and repeat revascularization were the secondary efficacy outcomes. Safety endpoints included contrast-associated acute kidney injury, stroke, major bleeding and stent thrombosis. All the endpoints were defined as reported in each study .2 statistic, and I2 > 50% was defined as statistical heterogeneity. For the primary endpoint, separate analyses were conducted according to the conditions described below: (1) trials with low risk of bias according to the Cochrane Collaboration's tool; (2) trials published in full text; (3) trials published after 2012; and (4) trials in that PCI of non-culprit vessels were mainly guided by angiography. Moreover, meta-regression analyses were carried out to assess the correlation of the following covariates with cardiovascular mortality or MI: age, gender, diabetes, hypertension, anterior MI, three-vessel disease, use of drug-eluting stent (DES) and duration of follow-up. To test whether the results were already definitive or may be implicitly influenced by novel data in the future, we conducted trial sequential analysis of the included RCTs for the primary endpoint, and the detailed methods were previously described  with 95% confidence intervals (CIs) were calculated using the Dersimonian and Lair random-effects model. Analysis was performed on intention-to-treat basis. Potential heterogeneity among studies was quantified with Iescribed .Network meta-analyses with a Bayesian framework were performed to combine both direct and indirect evidence about the 3 PCI strategies. Bayesian hierarchical random-effects models with directed acyclic graph models for general-purpose Markov chain Monte Carlo analysis were created with 20,000 tuning iterations and 50,000 simulation iterations. Convergence was assessed with the Brooks-Gelman-Rubin method and by visual inspection of convergence plots. Inconsistency was tested by contrasting direct with indirect evidence through node-splitting analysis. Of note, consistency model was utilized to draw conclusions, or an inconsistency model was adopted when statistical inconsistency was detected. The rank probability plot produced by the network meta-analysis was applied to present the best strategy. The risk of potential publication bias was assessed by visual inspection of funnel plots.p values were two-sided, and results were considered statistically significant at P < 0.05. Computations were performed using Review Manager 5.3 , ADDIS  and the trial sequential analysis software version 0.9.5.10 Beta.This study was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement . All p vEleven studies involving 6,942 patients were included in the final analysis , 23–26. The main characteristics of the eligible studies are presented in The baseline characteristics of the patients are detailed in 2 = 0%; and RR 0.73, 95% CI 0.61–0.88, I2 = 0%, respectively). However, the incidence of cardiovascular death or MI was not statistically different in staged and immediate complete revascularization groups   . Trial sI2 = 0%) .2 = 0%). However, no statistical differences were found when comparing staged complete revascularization with culprit-only PCI  or immediate complete revascularization  with respect to MI (2 = 0%) or as a staged procedure  compared with culprit-only PCI. No difference was found between staged complete revascularization and immediate complete revascularization groups with regard to repeat revascularization  (As shown in ct to MI . Repeat I2 = 0%) . In addiI2 = 0%) –S10.Separate analyses according to the pre-specified variables obtained mostly similar results compared to the overall analysis for all the comparisons . Meta-reWe did not find any inconsistencies between evidence from direct and indirect comparisons for all the efficacy endpoints, and the consistency model was consistently applied . NetworkThis comprehensive meta-analysis of 11 RCTs with 6,942 patients evaluated the relative efficacy and safety of the 3 PCI strategies for hemodynamically stable patients with STEMI and multivessel disease. The principal findings are as follows: (1) both immediate and staged complete revascularization significantly reduced the risks of cardiovascular death or MI and repeat revascularization compared with culprit-only PCI; (2) complete revascularization during primary PCI or as a staged procedure had similar incidence of safety outcomes compared with culprit-only PCI; and (3) No statistical difference was found between immediate and staged complete revascularization in terms of all the outcomes.The management of non-culprit vessels in STEMI has been the subject of extensive debate for 2 decades. In the past few years , several RCTs with moderate sample size highlighted lower risk of repeat revascularization or major adverse cardiovascular events, but not the hard endpoints in patients undergoing immediate or staged multivessel PCI compared with those undergoing culprit-only PCI –12. The Previous network meta-analysis conducted by Bangalore et al. showed that immediate complete revascularization might be the preferred strategy in patients with STEMI and multivessel disease due to the significantly reduced risks of all-cause death or MI, all-cause death, MI and repeat revascularization, whereas staged complete revascularization only reduced the risk of repeat revascularization, but did not reduce the risk of all-cause death or MI compared with culprit-only PCI . SimilarPotentially the most important finding of our study is that both immediate and staged complete revascularization can reduce the risk of cardiovascular death or MI compared with culprit-only PCI. One possible explanation of the beneficial effect of complete revascularization is that early PCI of non-culprit lesions can reduce ischemic burden, decrease infarct size and preserve left ventricular function, thereby reducing the risks of heart failure and cardiovascular mortality. In addition, pathophysiological changes such as pro-inflammatory state is generalized in all the arteries rather than in the culprit-vessel alone. Actually, results from the COMPLETE trial optical coherence tomography substudy indicated that 47% of patients who had at least one obstructive non-culprit lesion containing complex vulnerable plaque which were prone to major adverse cardiovascular events . TherefoMoreover, our meta-analysis based on current RCTs also found that immediate complete revascularization might be ranked as the best revascularization strategy followed by staged complete revascularization for the primary endpoint after the publication of the COMPLETE trial. Note that, staged complete revascularization was consistently not statistically different from immediate complete revascularization for all the outcomes with the currently limited sample size. In fact, the incidence of ischemic events is more common in the first days and then decreases after the first month . CompareIn previous guidelines, recommendations for revascularization of non-culprit vessels in STEMI patients were strongly influenced by prolonged intervention time, contrast-induced nephropathy, procedural complications and stent thrombosis in a pro-thrombotic and pro-inflammatory state . With thOur study presents several limitations that cannot be ignored. First, as a trial-level meta-analysis, this study was limited by variability in inclusion or exclusion criteria, procedural technique, stent type, timing of staged PCI, and follow-up duration. Therefore, random-effects model was used all across the study and sensitivity analyses yielded mostly similar results compared to the overall results. Furthermore, meta-regression analyses did not find any significant factor that contributed to the heterogeneity. Second, the results should be extrapolated to high-risk patients carefully. All RCTs included only hemodynamically stable patients, and patients with cardiogenic shock, left main disease, and CTO in non-culprit vessels were specifically excluded in most of the trials. Third, our study did not have enough data to suggest the optimal timing of staged complete revascularization, i.e., during hospitalization or within a few weeks after STEMI. Fourth, we did not evaluate whether the determination of non-culprit lesions requiring PCI should be guided by angiography or FFR, as FFR was mainly applied in only 3 trials. The ongoing trials will contribute to specify the better approach to guide the revascularization of non-culprit vessels . Fifth, blinding was not possible for the included trials, the knowledge that patients had untreated significant non-culprit lesions may by itself drive the incidence of new revascularizations in culprit-only PCI group.The current evidence suggests that complete revascularization during primary PCI or as a staged procedure is safe and feasible for hemodynamically stable patients with STEMI and multivessel disease. Both immediate and staged complete revascularization were associated with a reduction of cardiovascular death or MI compared with culprit-only PCI. According to trial sequential analysis, further trials are warranted to directly compare immediate vs. staged complete revascularization in terms of cardiovascular mortality or MI.The original contributions presented in the study are included in the article/KD and KC contributed to the study design and interpretation of the results. DY, LF, CZ, SY, and SW collected the data. KC, DY, and LF analyzed the data. KC prepared the manuscript. LF, KD, DY, and CZ revised the manuscript. All authors contributed to the article and approved the submitted version.The current study was funded by CAMS Innovation Fund for Medical Sciences (CIFMS) (2021-I2M-1-008) and Beijing Municipal Health Commission-Capital Health Development Research Project (2020-1-4032).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The treatment landscape of B-cell lymphomas is evolving with the advent of novel agents including immune and cellular therapies. Bispecific antibodies (bsAbs) are molecules that recognise two different antigens and are used to engage effector cells, such as T-cells, to kill malignant B-cells. Several bispecific antibodies have entered early phase clinical development since the approval of the CD19/CD3 bispecific antibody, blinatumomab, for relapsed/refractory acute lymphoblastic leukaemia. Novel bsAbs include CD20/CD3 antibodies that are being investigated in both aggressive and indolent non-Hodgkin lymphoma with encouraging overall response rates including complete remissions. These results are seen even in heavily pre-treated patient populations such as those who have relapsed after chimeric antigen receptor T-cell therapy. Potential toxicities include cytokine release syndrome, neurotoxicity and tumour flare, with a number of strategies existing to mitigate these risks. Here, we review the development of bsAbs, their mechanism of action and the different types of bsAbs and how they differ in structure. We will present the currently available data from clinical trials regarding response rates, progression free survival and outcomes across a range of non-Hodgkin lymphoma subtypes. Finally, we will discuss the key toxicities of bsAbs, their rates and management of these adverse events. In the last decade, novel therapies have emerged that can achieve complete remissions with durable response in patients with heavily pre-treated, relapsed/refractory (R/R) B-cell lymphomas, such as diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Immune cellular therapies such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies (bsAbs) have provided specificity to immuno-oncology with different methods to engage the patient’s own T-cells to kill malignant B-cells. Ongoing bsAb trials have expanded beyond the relapsed/refractory setting. In the upfront DLBCL setting, bsAbs are being combined with R-CHOP chemotherapy  as well as with agents such as polatuzumab vedotin, lenalidomide and atezolizumab. These strategies may lead to combination therapies that obviate the need for initial intensive chemotherapy in some non-Hodgkin lymphoma (NHL) subtypes. In an elderly population with co-morbidities, this may become an attractive alternative to the current standard of care of chemotherapy. However, bsAbs may present additional overlapping toxicities in addition to unique challenges, such as cytokine release syndrome (CRS), which must be mitigated to maximise their clinical efficacy.This review aims to highlight the different bsAbs currently in advanced stage clinical development for B-cell lymphomas. We will present their biology and mechanism of action, clinical efficacy data available to date and outline common toxicities and their management. Familiarity with these agents is important as they potentially represent a new drug class with a range of indications in the treatment of B-cell lymphomas. However, their treatment protocols can be complex and we aim to illustrate strategies to mitigate and manage key toxicities such as CRS.Bispecific antibodies have only recently become approved for use in haematological conditions with the clinical translation and efficacy of blinatumomab for R/R acute lymphoblastic leukaemia (ALL) and emicizumab for haemophilia A. However, the origin of the technology to create bsAbs can be traced back 50 years earlier when bispecific antigen binding fragments were successfully coupled and demonstrated agglutination of two different cell lines in vitro . The firWhile there are in excess of 100 academic and industry-derived molecules in clinical development, many with similar antigen/receptor targets, there are vast differences between the molecules in their manufacturing and structure. There are mechanistic differences in the activity of bsAbs, with the majority in advanced clinical development being designed to facilitate T-cell engagement and activation of antibody-dependent cellular cytotoxicity (ADCC) against the tumour cell ultimately via perforin/granzyme-mediated cell death. This is achieved through direct binding of CD3ε of the T-cell receptor complex (bypassing T-cell major histocompatibility complex specificity) to a cell surface tumour antigen on the target cell . Other mSimilar to naturally occurring antibodies, bsAbs involve permutations of heavy and light chain variable domains, with or without retention of the fragment crystallisable region (Fc) domain. The Fc domain is the tail region of the antibody which interacts with cell surface receptors, Fc receptors. The antigen-binding fragment (Fab) is the part of the antibody that binds to the antigen. As a naturally occurring antibody contains two heavy and two light chain variable regions, there can be 16 potential recombinations resulting in significant antibody diversity during manufacturing. One method to overcome this is to generate individual antigen-binding fragments (Fabs) and fuse them via chemical coupling such as oxidation or physical linkage through addition of linker proteins. Blinatumomab is an example of a fragment based bsAb, wherein antibody fragments are combined without the presence of the Fc domain. While this structure is easier and cheaper to manufacture, the absence of the Fc region is the reason that the molecule undergoes rapid degradation. These unfavourable pharmacokinetics explain its requirement for constant intravenous infusion and also its limited clinical activity in the treatment of NHL as compared with ALL.The majority of bsAbs in advanced stage clinical development for lymphoma are those containing the Fc region, as these most closely resemble naturally occurring IgG antibodies and hence maintain favourable pharmacokinetic properties. This also renders these bsAbs to be least immunogenic, enhancing tolerability and efficacy upon repeat administration. The Fc domain of bsAbs is commonly re-engineered to prevent FcyRs and C1q binding to prevent excessive cytokine release from recruited T cells and to minimise off-target toxicity such as T-cell mediated hepatotoxicity. Preserving FcRn binding prolongs the half-life of the agent in vivo. The knob and hole manufacturing approach involves separately derived half antibodies with “knob” and “hole” mutations in the CH3 domains that facilitate homodimerization . ReductiAnother key difference between certain bsAbs is the number of Fab arms within each antibody. Mosunetuzumab  has a 1:1 CD3:CD20 ratio of Fab arms, whereas glofitamab  has a 1:2 CD3:CD20 ratio, facilitating higher avidity of the binding of the T-cell via two CD20 antigens on the target B-cell simultaneously. Notably, the CD20 Fab of glofitamab is derived from the 2nd generation CD20 monoclonal antibody, obinutuzumab, which itself was glycoengineered to enhance binding and increase ADCC compared to first generation monoclonal CD20 Abs such as rituximab. It is hypothesised that the differential avidity of the antibodies due to the differing CD3:CD20 ratios is the reason for possible differential activity of these molecules against aggressive and indolent B-cell histologies, although this observation is subject to further preclinical and clinical investigation.In contrast to other bsAbs based around an IgG backbone, IGM-2323  is a bsAb based on an IgM backbone with grafting of IgG antibodies onto the multimeric IgM frame and addition of a CD3-binding Fv domain . This crWhile the majority of molecules in advanced clinical development are CD3/CD20 bsAbs, early phase clinical translation includes a myriad of other effector targets and mechanisms. RG6076  is an example of alternative B- and T-cell antigenic targets in order to engage T-cell ADCC. AFM13 (CD16A/CD30) engages NK-cells and macrophages via CD16A to CD30+ malignancies such as CD30+ T-cell lymphomas and Hodgkin lymphoma. Indeed, as the efficiency of design and production has developed, bispecific antibodies can be created with virtually any desired antigen specificities for malignant and non-malignant indications. Of B-cell malignancies, bsAbs were first used to treat R/R B-ALL. Blinatumomab, an anti-CD19/CD3 bispecific antibody, showed efficacy in B-ALL and was approved by the Food and Drug Administration (FDA) for this indication in 2014 ,9. On th2/day achieved poor response rates and a dose of 90 µg/m2/day was limited by neurotoxicity. In the extension phase, 11 patients with R/R DLBCL received the target dose with an overall response rate (ORR) of 55% and a complete remission (CR)/unconfirmed complete remission rate of 36% .,19.11,19CRS is a common systemic inflammatory response seen in response to antibody and adoptive T-cell therapies, resulting from immune activation and release of inflammatory cytokines . IL-6 isCRS is the most common adverse event seen in many bsAbs used for the treatment of R/R NHL . In a phFever within 24 h is the hallmark symptom, with significant variability in other aspects of the clinical presentation. Mild features include fatigue, headache, rash, arthralgia and myalgia. Severe CRS is a medical emergency and can present with circulatory shock, respiratory dysfunction, neurotoxicity and disseminated intravascular coagulation . IntercuA number of disparate grading systems for CRS have been developed ,34,35,36Patient, disease and therapy-related factors influence the risk of developing severe CRS. Patients with high disease burden are more likely to experience severe CRS ,38,39,40Mitigation strategies to reduce severe CRS are commonly used with bsAbs. Premedications are regularly employed with steroids, antihistamines and acetaminophen or paracetamol . Step-upManagement of CRS follows a grade-adapted strategy. Mild grade 1 CRS is managed with supportive therapy including infusion discontinuation, intravenous fluids, antipyretics and antihistamines. Prolonged or severe CRS requires close monitoring of organ function, generally within an intensive care unit. Tocilizumab, a humanized, IgG, anti-human IL-6R monoclonal antibody is the mainstay of treatment for grade 2 or higher CRS . It is eTocilizumab does not efficiently penetrate the blood–brain barrier and so corticosteroids are used in cases with significant neurotoxicity or co-existing ICANS . OtherwiICANS is a pathological process involving the central nervous system (CNS) following immunotherapy that activates endogenous or infused T-cells . ICANS iNeurotoxicity is common with blinatumomab, occurring in 70% of R/R NHL patients in a phase 2 trial, including 22% with grade 3 or higher toxicity . The toxICANS generally develops within the first seven days of therapy . HeadachSimilar to CRS, a number of different ICANS grading systems have been developed ,36. The Many of the risk factors for CRS also apply to ICANS. Early or severe CRS increases the risk of developing ICANS. High tumour burden, thrombocytopenia, high ferritin and pre-existing neurological co-morbidities are also risk factors ,56,57. MManagement of ICANS involves thorough and serial investigations with EEG, fundoscopic examination and neuroimaging to exclude other causes of neurotoxicity and to identify further complications. Supportive measures are essential, including elevation of the bed to at least 30°, aspiration precautions, avoiding other CNS depressing medications and mechanical ventilation for airway protection if required. Pharmacological treatment is with high dose corticosteroids  followed by a tapered course. Tocilizumab is not used for ICANS management due to its limited ability to cross the blood-brain-barrier, although it is often administered due to the common co-occurrence with CRS .Tumour flare is a well described immune reaction in NHL, particularly with exposure to immunomodulators such as thalidomide and lenalidomide ,60. A siDose-limiting tumour flare has been reported with glofitamab when used in combination with atezolizumab, an anti-PD-L1 inhibitor . ProphylSince the approval of blinatumomab for R/R ALL, there has been significant investment into the development of bsAbs across the spectrum of indolent to aggressive lymphomas. The off-the-shelf preparation to provide cell-specific immune engagement has provided a new therapeutic option and, based on early clinical development described in this review, combinatorial and monotherapy bsAb therapy is being tested from frontline through to heavily pre-treated patients, including those who progressed after CAR T-cell therapy.This review has highlighted some of the mechanistic differences between bsAbs in advanced stage clinical development for lymphoma. Preclinical and biomarker studies including pharmacokinetics and cytokine assays will assist understanding as to the different treatment responses, tumour kinetics and AE profiles of bsAbs. The AE profile is distinct from non-cellular therapies, but relatively uniform to the class and once familiarity with CRS identification, classification and management is achieved, clinicians are well poised to implement the use of bsAbs into widespread clinical practice as safety and efficacy endpoints are met and the likely pathway to approval achieved.


Variations in COVID-19 prevalence, severity, and mortality rate remain ambiguous. Genetic or individual differences in immune response may be an explanation. Moreover, hyperinflammation and dysregulated immune response are involved in the etiology of severe forms of COVID-19. Therefore, the aim of the present study was to analyze serum alpha-1 antitrypsin (AAT) levels, as an acute-phase plasma protein with immunomodulatory effect and neutrophil to lymphocyte ratio (NLR) as a marker of inflammation response in severe COVID-19 illness.  In this retrospective observational cohort study, 64 polymerase chain reaction (PCR) positive COVID-19 hospitalized patients were studied for AAT, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), troponin, complete blood count (CBC), random blood sugar, serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), and arterial oxygen saturation (O2sat) at admission and during hospitalization. p < 0.05) and troponin levels (p < 0.05). However, comorbidities, CRP alterations, ESR alterations, nonfasting blood sugar, SGOT, SGPT, O2sat, RBC, and PLT values were not significantly different between the NLR downward and upward trend groups.  The results showed that hospitalized patients with COVID-19 had low serum levels of AAT and high CRP levels at the first days of hospitalization. In particular, the percentages of individuals with low, normal, and high AAT levels were 7.80%, 82.80%, and 9.40%, respectively, while high and low values of CRP accounted for 86.70% and 13.30% of patients. Most of the patients had an upward neutrophil to lymphocyte ratio (NLR) trend, with a higher mortality rate ( The current study revealed that severe COVID-19 patients had low serum AAT levels related to CRP values. Therefore, AAT response may be considered as a new mechanism by which some COVID-19 patients show immune dysregulation and more severe symptoms. Since the end of 2019, the COVID-19 outbreak has spread almost all over the world at an exponential rate. Although the presentation of vaccines in early 2021 may decrease the rate of studies in COVID-19 treatments, new variants of SARS-CoV-2 with a high potency of escaping conventional vaccines motivate us to search for risk factors involved in the occurrence of severe cases of the disease.The clinical manifestations of COVID-19 vary from asymptomatic and mild to severe forms. Some evidence has been estimated that the severe form of COVID-19 threatens 1.7 billion people (1 in 5) around the world due to at least one risk factor .κB) [The main targets of SARS-CoV-2 for cell entry are angiotensin-converting enzyme 2 (ACE2) and some serine proteases such as transmembrane serine protease 2 (TMPRSS2). After virus entrance, intracellular antiviral mechanisms lead to the secretion of interferons (IFNs) following the recognition of virus components by host pattern recognition receptors (PRRs), which trigger an inflammatory response by activation of nuclear factor kappa B (NFκB) .Inflammation is the main part of the host response to any pathogen; however, patients with COVID-19, particularly in the severe form of the disease, catastrophically show signs of acute hyperinflammation and produce systemic markers of inflammation such as CRP, high levels of white blood cells, and proinflammatory cytokines . The sigOne of the main findings of severe COVID-19 is the increase in neutrophil (neutrophilia) and decrease in lymphocyte (lymphopenia) counts (high NLR). These symptoms reveal a dysregulated form of the immune response as a result of highly activated innate immunity . In partIf the role of dysregulated immune response and increase of NLR in the pathogenesis of severe COVID-19 illness are accepted in an algorithmic approach, it is plausible to consider the natural immune-modulatory mechanisms and probable impairment of these mechanisms in some people.SERPINA-1 gene and expressed as a 51 kD glycoprotein [SERPINA-1 gene lead to hepatic protein synthesis defection and AAT deficiency (AATD) [AAT is first recognized as an antiprotease that mainly releases from the liver and inhibits proteases derived from activated neutrophils, particularly those infiltrated in the lungs. AAT is encoded by the oprotein . In addioprotein . Some otoprotein . AAT wity (AATD) . Individy (AATD) . The maiThis retrospective observational cohort study was performed on 64 consecutive hospitalized COVID-19 patients who were admitted to the hospitals affiliated to Shahid Beheshti University of Medical Sciences from July 2020 to November 2020 . PatientClinical and biological characteristics of the patients were obtained from the hospital's electronic medical records. Data were recorded by data entry teams into a computerized database and reviewed by two experienced clinicians. Recorded comorbidities included any type of cancer, type 2 diabetes mellitus, and heart diseases .AAT was determined by an immunoturbidimetric assay using a Roche COBAS INTEGRA 400 plus analyzer  with 20 mg/dl sensitivity. Red blood cells (RBC), platelets, neutrophil, and lymphocyte counts and their percentages were analyzed automatically by a blood cell counter . CRP, random blood sugar (BS), SGOT, and SGPT were measured by using the diagnostic kits from Pars Azmoon Company  and an autoanalyzer Selectra ProXL . The kits used in this study had a sensitivity of 5 mg/dl for BS, 2 IU/l for SGOT, and 4 IU/l for SGPT. Measurement of ESR was made using the Westergren method and ESR tube according to standard procedure. Troponin concentration was examined by an enzyme-linked immunoabsorbent assay (ELISA) kit . The NLR value was calculated by dividing the neutrophil counts by the lymphocyte counts. O2sat was measured directly by an ABL90 FLEX PLUS blood gas analyzer . Blood samples were taken at admission and during hospitalization. Only the first and last test results of neutrophil and lymphocyte counts, CRP, ESR, and NLR were recorded.The primary aim of this study was to examine whether the serum level of AAT reduces in the COVID-19 patients. Moreover, the study focused on two patient subtypes: cases with NLR decreasing trend and those with NLR increasing trend during the study. Differences in patients' clinical and laboratory characteristics were evaluated between the two subtypes.p value < 0.05 was considered statistically significant. Continuous variables were presented as means ± SD or medians with interquartile ranges (IQRs), based on the normal or skewed distribution. Categorical variables were presented as number (%). The independent-samples t-test for normally distributed variables and the Mann-Whitney U test for nonnormally distributed data were used. For categorical variables, the chi-square (χ2) test and Fisher's exact or Fisher-Freeman-Halton test were applied [Statistical analyses were performed by using SPSS software, version 20. A two-tailed  applied .This study includes 64 severe COVID-19 patients, who were mostly hospitalized in the ICU. However, few cases were excluded due to some missing parameters, later on . They wep < 0.05). Comorbidities were not significantly different between NLR downward and upward trend groups.The participants were subdivided into two groups according to increased or decreased neutrophil to lymphocyte ratio during hospitalization. The overall mortality rate was 41%, significantly different between NLR upward (51%) and downward (21%) groups . Nonfasting blood sugar in severe COVID-19 patients was higher than the normal range (less than 140 mg/dl), though the glucose levels were not significantly different between the NLR groups. Liver enzymes, SGOT, and SGPT also encountered moderate changes in many patients compared to the normal range. However, this increase was not different between NLR upward and downward trend groups. RBC and PLT values were also not significant between the two groups. Moreover, O2sat was slightly higher in the NLR downward trend group, although the difference between the two groups was not significant.Troponin was another marker that increased in most patients. The serum level of this cardiac biomarker was significantly higher in the NLR upward trend group (Finally, the current study results showed that 82.80% of patients had normal AAT levels. The percentages of individuals with low and high AAT levels were 7.80% and 9.40%, respectively . MoreoveThe results of the present study showed that severe COVID-19 patients had serum AAT levels mostly in the normal range at the time of admission, despite high baseline levels of CRP and ESR. The patients who showed an increasing NLR trend during hospital attendance had a higher mortality rate and slightly lower, but not significant, serum AAT levels than patients with decreasing NLR trend. Participants with the upward NLR trend had also higher serum troponin levels. The baseline levels of CRP, ESR, and their alterations during hospitalization were not significantly different between increasing and decreasing NLR trend groups.We observed 41% mortality in patients diagnosed as severe COVID-19. In a Mexico City study, 31% of patients admitted to hospital had died as severe COVID-19 . This diPI∗ZZ, PI∗SZ, and PI∗MZ genotypes compared to other parts of Europe and the world. Interestingly, the mortality rate of these countries from severe COVID-19 cases was the highest among other countries [AAT is an acute-phase plasma protein that increases by 75 to 100% during inflammation accompanied by other acute-phase proteins such as CRP or ESR. Compared to baseline levels, the inflammation-induced elevation of AAT is observed in the individuals with homozygote and heterozygote forms of AATD. Therefore, normal levels of AAT in AATD may be due to the inflammation that induces elevation of lower baseline. It has been shown that CRP is not influenced by AATD; therefore, AAT should be considered in correlation with CRP to better estimate AAT response in different genotypes of AAT during normal and inflammatory states . CRP levountries , 16. Viaountries . Besidesountries . Mountinountries .The elevation of NLR is due to increased neutrophils or decreased lymphocyte counts. NLR has potential utility in clinical applications for the prognosis of inflammatory diseases including ischemic stroke , cerebraThe recent evidence indicates that SARS-CoV-2 had an adverse effect on the cardiovascular system, and many patients treated for COVID-19 may face thrombosis and heart attack, a few months after treatment . We obseIn conclusion, the results of the present study demonstrate that severe COVID-19 patients may release inadequate amount of AAT proteins in the blood circulation and thus encounter neutrophilia and lymphopenia. Therefore, AAT response may be considered as a new mechanism by which some COVID-19 patients show dysregulated inflammation and more severe symptoms. Since AAT-rich plasma is administered to individuals with AATD and some other conditions such as diabetes type 1 and influenza, it is plausible to consider it as a new treatment for severe COVID-19 patients.