First Affiliated Hospital of Jinan University

autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

BACKGROUND: Stroke is common during the first few weeks after a transient ischemic attack (TIA) or minor ischemic stroke. Combination therapy with clopidogrel and aspirin may provide greater protection against subsequent stroke than aspirin alone. METHODS: In a randomized, double-blind, placebo-controlled trial conducted at 114 centers in China, we randomly assigned 5170 patients within 24 hours after the onset of minor ischemic stroke or high-risk TIA to combination therapy with clopidogrel and aspirin (clopidogrel at an initial dose of 300 mg, followed by 75 mg per day for 90 days, plus aspirin at a dose of 75 mg per day for the first 21 days) or to placebo plus aspirin (75 mg per day for 90 days). All participants received open-label aspirin at a clinician-determined dose of 75 to 300 mg on day 1. The primary outcome was stroke (ischemic or hemorrhagic) during 90 days of follow-up in an intention-to-treat analysis. Treatment differences were assessed with the use of a Cox proportional-hazards model, with study center as a random effect. RESULTS: Stroke occurred in 8.2% of patients in the clopidogrel-aspirin group, as compared with 11.7% of those in the aspirin group (hazard ratio, 0.68; 95% confidence interval, 0.57 to 0.81; P<0.001). Moderate or severe hemorrhage occurred in seven patients (0.3%) in the clopidogrel-aspirin group and in eight (0.3%) in the aspirin group (P=0.73); the rate of hemorrhagic stroke was 0.3% in each group. CONCLUSIONS: Among patients with TIA or minor stroke who can be treated within 24 hours after the onset of symptoms, the combination of clopidogrel and aspirin is superior to aspirin alone for reducing the risk of stroke in the first 90 days and does not increase the risk of hemorrhage. (Funded by the Ministry of Science and Technology of the People's Republic of China; CHANCE ClinicalTrials.gov number, NCT00979589.).

Coronavirus disease 2019 (COVID-19), which began in Wuhan, China, in December 2019, has caused a large global pandemic and poses a serious threat to public health. More than 4 million cases of COVID-19, which is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have been confirmed as of 11 May 2020. SARS-CoV-2 is a highly pathogenic and transmissible coronavirus that primarily spreads through respiratory droplets and close contact. A growing body of clinical data suggests that a cytokine storm is associated with COVID-19 severity and is also a crucial cause of death from COVID-19. In the absence of antivirals and vaccines for COVID-19, there is an urgent need to understand the cytokine storm in COVID-19. Here, we have reviewed the current understanding of the features of SARS-CoV-2 and the pathological features, pathophysiological mechanisms, and treatments of the cytokine storm induced by COVID-19. In addition, we suggest that the identification and treatment of the cytokine storm are important components for rescuing patients with severe COVID-19.

Abstract In this study, we investigated lipopolysaccharide (LPS)-induced cognitive impairment and neuroinflammation in C57BL/6J mice by using behavioral tests, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and Western blot. We found that LPS treatment leads to sickness behavior and cognitive impairment in mice as shown in the Morris water maze and passive avoidance test, and these effects were accompanied by microglia activation (labeled by ionized calcium binding adaptor molecule-1, IBA-1) and neuronal cell loss (labeled by microtubule-associated protein 2, MAP-2) in the hippocampus. The levels of interleukin-4 (IL-4) and interleukin-10 (IL-10) in the serum and brain homogenates were reduced by the LPS treatment, while the levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), prostaglandin E2 (PGE 2 ) and nitric oxide (NO) were increased. In addition, LPS promoted the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in the brain homogenates. The Western blot analysis showed that the nuclear factor kappa B (NF-κB) signaling pathway was activated in the LPS groups. Furthermore, VIPER, which is a TLR-4-specific inhibitory peptide, prevented the LPS-induced neuroinflammation and cognitive impairment. These data suggest that LPS induced cognitive impairment and neuroinflammation via microglia activation by activating the NF-kB signaling pathway; furthermore, we compared the time points, doses, methods and outcomes of LPS administration between intraperitoneal and intracerebroventricular injections of LPS in LPS-induced neuroinflammation and cognitive impairment, and these data may provide additional insight for researchers performing neuroinflammation research.

Major depressive disorder (MDD) is common and disabling, but its neuropathophysiology remains unclear. Most studies of functional brain networks in MDD have had limited statistical power and data analysis approaches have varied widely. The REST-meta-MDD Project of resting-state fMRI (R-fMRI) addresses these issues. Twenty-five research groups in China established the REST-meta-MDD Consortium by contributing R-fMRI data from 1,300 patients with MDD and 1,128 normal controls (NCs). Data were preprocessed locally with a standardized protocol before aggregated group analyses. We focused on functional connectivity (FC) within the default mode network (DMN), frequently reported to be increased in MDD. Instead, we found decreased DMN FC when we compared 848 patients with MDD to 794 NCs from 17 sites after data exclusion. We found FC reduction only in recurrent MDD, not in first-episode drug-naïve MDD. Decreased DMN FC was associated with medication usage but not with MDD duration. DMN FC was also positively related to symptom severity but only in recurrent MDD. Exploratory analyses also revealed alterations in FC of visual, sensory-motor, and dorsal attention networks in MDD. We confirmed the key role of DMN in MDD but found reduced rather than increased FC within the DMN. Future studies should test whether decreased DMN FC mediates response to treatment. All R-fMRI indices of data contributed by the REST-meta-MDD consortium are being shared publicly via the R-fMRI Maps Project.

Given the increasing prevalence of diabetes and obesity worldwide, the deleterious effects of non-alcoholic fatty liver disease (NAFLD) are becoming a growing challenge for public health. NAFLD is the most common chronic liver disease in the Western world. NAFLD is closely associated with metabolic disorders, including central obesity, dyslipidaemia, hypertension, hyperglycaemia and persistent abnormalities of liver function tests.In general NAFLD is a common denominer for a broad spectrum of damage to the liver, which can be due to hepatocyte injury, inflammatory processes and fibrosis. This is normally seen on liver biopsy and can range from milder forms (steatosis) to the more severe forms (non-alcoholic steatohepatitis (NASH), advanced fibrosis, cirrhosis and liver failure). In these patients, advanced fibrosis is the major predictor of morbidity and liver-related mortality, and an accurate diagnosis of NASH and NAFLD is mandatory. Histologic evaluation with liver biopsy remains the gold standard to diagnose NAFLD. Diagnosis of NAFLD is defined as presence of hepatic steatosis, ballooning and lobular inflammation with or without fibrosis. Weight loss, dietary modification, and the treatment of underlying metabolic syndrome remain the mainstays of therapy once the diagnosis is established. Dietary recommendations and lifestyle interventions, weight loss, and the treatment of underlying metabolic syndrome remain the mainstays of therapy once the diagnosis is established with promising results but are difficult to maintain. Pioglitazone and vitamin E are recommended by guidelines in selected patients. This review gives an overview of NAFLD and its treatment options.

Due to their unique physicochemical properties, graphene-family nanomaterials (GFNs) are widely used in many fields, especially in biomedical applications. Currently, many studies have investigated the biocompatibility and toxicity of GFNs in vivo and in intro. Generally, GFNs may exert different degrees of toxicity in animals or cell models by following with different administration routes and penetrating through physiological barriers, subsequently being distributed in tissues or located in cells, eventually being excreted out of the bodies. This review collects studies on the toxic effects of GFNs in several organs and cell models. We also point out that various factors determine the toxicity of GFNs including the lateral size, surface structure, functionalization, charge, impurities, aggregations, and corona effect ect. In addition, several typical mechanisms underlying GFN toxicity have been revealed, for instance, physical destruction, oxidative stress, DNA damage, inflammatory response, apoptosis, autophagy, and necrosis. In these mechanisms, (toll-like receptors-) TLR-, transforming growth factor β- (TGF-β-) and tumor necrosis factor-alpha (TNF-α) dependent-pathways are involved in the signalling pathway network, and oxidative stress plays a crucial role in these pathways. In this review, we summarize the available information on regulating factors and the mechanisms of GFNs toxicity, and propose some challenges and suggestions for further investigations of GFNs, with the aim of completing the toxicology mechanisms, and providing suggestions to improve the biological safety of GFNs and facilitate their wide application.

RATIONALE: The prevalence of chronic obstructive pulmonary disease (COPD) in China is largely unknown. OBJECTIVES: To obtain the COPD prevalence in China through a large-population, spirometry-based, cross-sectional survey of COPD. METHODS: Urban and rural population-based cluster samples were randomly selected from seven provinces/cities. All residents 40 years of age or older in the selected clusters were interviewed with a standardized questionnaire revised from the international BOLD (Burden of Obstructive Lung Diseases) study. Spirometry was performed on all eligible participants. Patients with airflow limitation (FEV(1)/FVC < 0.70) were further examined by post-bronchodilator spirometry, chest radiograph, and electrocardiogram. Post-bronchodilator FEV(1)/FVC of less than 70% was defined as the diagnostic criterion of COPD. MEASUREMENTS AND MAIN RESULTS: Among 25,627 sampling subjects, 20,245 participants completed the questionnaire and spirometry (response rate, 79.0%). The overall prevalence of COPD was 8.2% (men, 12.4%; women, 5.1%). The prevalence of COPD was significantly higher in rural residents, elderly patients, smokers, in those with lower body mass index, less education, and poor ventilation in the kitchen, in those who were exposed to occupational dusts or biomass fuels, and in those with pulmonary problems in childhood and family history of pulmonary diseases. Among the patients who had COPD, 35.3% were asymptomatic; only 35.1% reported lifetime diagnosis of bronchitis, emphysema, or other COPD; and only 6.5% have been tested with spirometry. CONCLUSIONS: COPD is prevalent in individuals 40 years of age or older in China.

BACKGROUND: Altered composition of the gut microbiota is involved in both the onset and progression of obesity and diabetes mellitus. However, the link between gut microbiota and obesity-related cardiovascular complications has not been explored. The present study was designed to investigate the role of Akkermansia muciniphila, a mucin-degrading bacterium with beneficial effects on metabolism, in the pathogenesis of atherosclerosis in apolipoprotein E-deficient (Apoe(-/-)) mice. METHODS AND RESULTS: Apoe(-/-) mice on normal chow diet or a Western diet were treated with A muciniphila by daily oral gavage for 8 weeks, followed by histological evaluations of atherosclerotic lesion in aorta. Real-time polymerase chain reaction analysis demonstrated that the fecal abundance of A muciniphila was significantly reduced by Western diet. Replenishment with A muciniphila reversed Western diet-induced exacerbation of atherosclerotic lesion formation without affecting hypercholesterolemia. A muciniphila prevented Western diet-induced inflammation in both the circulation and local atherosclerotic lesion, as evidenced by reduced macrophage infiltration and expression of proinflammatory cytokines and chemokines. These changes were accompanied by a marked attenuation in metabolic endotoxemia. A muciniphila-mediated reduction in circulating endotoxin level could be attributed to the induction of intestinal expression of the tight junction proteins (zona occuldens protein-1 and occludin), thereby reversing Western diet-induced increases in gut permeability. Long-term infusion of endotoxin to Apoe(-/-) mice reversed the protective effect of A muciniphila against atherosclerosis. CONCLUSION: A muciniphila attenuates atherosclerotic lesions by ameliorating metabolic endotoxemia-induced inflammation through restoration of the gut barrier.

The widespread expression of circular RNAs (circRNAs) is regarded as a feature of gene expression in highly diverged eukaryotes. Recent studies have shown that circRNAs can act as a miRNA sponge to repress miRNA function, participate in splicing of target genes, translate genes into protein and interact with RNA binding proteins (RBPs). RBPs are a broad class of proteins involved in gene transcription and translation, and interaction with RBPs is considered an important part of circRNA function, which can serve as an essential element underlying the functions of circRNAs, including genesis, translation, transcriptional regulation of target genes, and extracellular transport. In this mini-review, we attempt to explore in detail the relationship between circRNAs and RBPs, and the interactions between the two factors. The goal of this review is to investigate the emerging studies of RBPs and circRNAs to better understand how their interaction alters cellular function.

BACKGROUND AND PURPOSE: We aimed to establish the prevalence, characteristics, and outcomes of intracranial atherosclerosis (ICAS) in China by a large, prospective, multicenter study. METHODS: We evaluated 2864 consecutive patients who experienced an acute cerebral ischemia<7 days after symptom onset in 22 Chinese hospitals. All patients underwent magnetic resonance angiography, with measurement of diameter of the main intracranial arteries. ICAS was defined as ≥50% diameter reduction on magnetic resonance angiography. RESULTS: The prevalence of ICAS was 46.6% (1335 patients, including 261 patients with coexisting extracranial carotid stenosis). Patients with ICAS had more severe stroke at admission and stayed longer in hospitals compared with those without intracranial stenosis (median National Institutes of Health Stroke Scale score, 3 versus 5; median length of stay, 14 versus 16 days; both P<0.0001). After 12 months, recurrent stroke occurred in 3.27% of patients with no stenosis, in 3.82% for those with 50% to 69% stenosis, in 5.16% for those with 70% to 99% stenosis, and in 7.27% for those with total occlusion. Cox proportional hazards regression analyses showed that the degree of arterial stenosis, age, family history of stroke, history of cerebral ischemia or heart disease, complete circle of Willis, and National Institutes of Health Stroke Scale score at admission were independent predictors for recurrent stroke at 1 year. The highest rate of recurrence was observed in patients with occlusion with the presence of ≥3 additional risk factors. CONCLUSIONS: ICAS is the most common vascular lesion in patients with cerebrovascular disease in China. Recurrent stroke rate in our study was lower compared with those of previous clinical trials but remains unacceptably high in a subgroup of patients with severe stenosis.

BACKGROUND: Ferroptosis is a newly recognized type of cell death, which is different from traditional necrosis, apoptosis or autophagic cell death. However, the position of ferroptosis in lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored intensively so far. In this study, we mainly analyzed the relationship between ferroptosis and LPS-induced ALI. METHODS: In this study, a human bronchial epithelial cell line, BEAS-2B, was treated with LPS and ferrostatin-1 (Fer-1, ferroptosis inhibitor). The cell viability was measured using CCK-8. Additionally, the levels of malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), and iron, as well as the protein level of SLC7A11 and GPX4, were measured in different groups. To further confirm the in vitro results, an ALI model was induced by LPS in mice, and the therapeutic action of Fer-1 and ferroptosis level in lung tissues were evaluated. RESULTS: The cell viability of BEAS-2B was down-regulated by LPS treatment, together with the ferroptosis markers SLC7A11 and GPX4, while the levels of MDA, 4-HNE and total iron were increased by LPS treatment in a dose-dependent manner, which could be rescued by Fer-1. The results of the in vivo experiment also indicated that Fer-1 exerted therapeutic action against LPS-induced ALI, and down-regulated the ferroptosis level in lung tissues. CONCLUSIONS: Our study indicated that ferroptosis has an important role in the progression of LPS-induced ALI, and ferroptosis may become a novel target in the treatment of ALI patients.

Data mining technology can search for potentially valuable knowledge from a large amount of data, mainly divided into data preparation and data mining, and expression and analysis of results. It is a mature information processing technology and applies database technology. Database technology is a software science that researches manages, and applies databases. The data in the database are processed and analyzed by studying the underlying theory and implementation methods of the structure, storage, design, management, and application of the database. We have introduced several databases and data mining techniques to help a wide range of clinical researchers better understand and apply database technology.

Relapse to anti-HER2 monoclonal antibody (mAb) therapies, such as trastuzumab in HER2 + breast cancer (BC), is associated with residual disease progression due to resistance to therapy. Here, we identify interferon-γ inducible protein 16 (IFI16)-dependent STING signaling as a significant determinant of trastuzumab responses in HER2 + BC. We show that down-regulation of immune-regulated genes (IRG) is specifically associated with poor survival of HER2 + , but not other BC subtypes. Among IRG, IFI16 is identified as a direct target of EZH2, the underexpression of which leads to deficient STING activation and downstream CXCL10/11 expression in response to trastuzumab treatment. Dual inhibition of EZH2 and histone deacetylase (HDAC) significantly activates IFI16-dependent immune responses to trastuzumab. Notably, a combination of a novel histone methylation inhibitor with an HDAC inhibitor induces complete tumor eradication and long-term T cell memory in a HER2 + BC mouse model. Our findings demonstrate an epigenetic regulatory mechanism suppressing the expression of the IFI16-CXCL10/11 signaling pathway that provides a survival advantage to HER2 + BC to confer resistance to trastuzumab treatment.

China faces the greatest challenge from stroke in the world. The death rate for cerebrovascular diseases in China was 149.49 per 100 000, accounting for 1.57 million deaths in 2018. It ranked third among the leading causes of death behind malignant tumours and heart disease. The age-standardised prevalence and incidence of stroke in 2013 were 1114.8 per 100 000 population and 246.8 per 100 000 person-years, respectively. According to the Global Burden of Disease Study 2017, the years of life lost (YLLs) per 100 000 population for stroke increased by 14.6%; YLLs due to stroke rose from third highest among all causes in 1990 to the highest in 2017. The absolute numbers and rates per 100 000 population for all-age disability-adjusted life years (DALYs) for stroke increased substantially between 1990 and 2017, and stroke was the leading cause of all-age DALYs in 2017. The main contributors to cerebrovascular diseases include behavioural risk factors (smoking and alcohol use) and pre-existing conditions (hypertension, diabetes mellitus, dyslipidaemia and atrial fibrillation (AF)). The most prevalent risk factors among stroke survivors were hypertension (63.0%-84.2%) and smoking (31.7%-47.6%). The least prevalent was AF (2.7%-7.4%). The prevalences for major risk factors for stroke are high and most have increased over time. Based on the latest national epidemiological data, 26.6% of adults aged ≥15 years (307.6 million adults) smoked tobacco products. For those aged ≥18 years, age-adjusted prevalence of hypertension was 25.2%; adjusted prevalence of hypercholesterolaemia was 5.8%; and the standardised prevalence of diabetes was 10.9%. For those aged ≥40 years, the standardised prevalence of AF was 2.31%. Data from the Hospital Quality Monitoring System showed that 3 010 204 inpatients with stroke were admitted to 1853 tertiary care hospitals during 2018. Of those, 2 466 785 (81.9%) were ischaemic strokes (ISs); 447 609 (14.9%) were intracerebral haemorrhages (ICHs); and 95 810 (3.2%) were subarachnoid haemorrhages (SAHs). The average age of patients admitted was 66 years old, and nearly 60% were male. A total of 1555 (0.1%), 2774 (0.6%) and 1347 (1.4%) paediatric strokes (age <18 years) were identified among IS, ICH and SAH, respectively. Over one-third (1 063 892 (35.3%)) of the patients were covered by urban resident basic medical insurance, followed by urban employee basic medical insurance (699 513 (23.2%)) and new rural cooperative medical schema (489 361 (16.3%)). The leading risk factor was hypertension (67.4% for IS, 77.2% for ICH and 49.1% for SAH), and the leading comorbidity was pneumonia or pulmonary infection (10.1% for IS, 31.4% for ICH and 25.2% for SAH). In-hospital death/discharge against medical advice rate was 8.3% for stroke inpatients, ranging from 5.8% for IS to 19.5% for ICH. The median and IQR of length of stay was 10.0 (7.0-14.0) days, ranging from 10.0 (7.0-13.0) in IS to 14.0 (8.0-22.0) in SAH. Data from the Chinese Stroke Center Alliance demonstrated that the composite scores of guideline-recommended key performance indicators for patients with IS, ICH and SAH were 0.77±0.21, 0.72±0.28 and 0.59±0.32, respectively.

Excessive inflammatory responses induced upon SARS-CoV-2 infection are associated with severe symptoms of COVID-19. Inflammasomes activated in response to SARS-CoV-2 infection are also associated with COVID-19 severity. Here, we show a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation to induce hyperinflammation. N protein facilitates maturation of proinflammatory cytokines and induces proinflammatory responses in cultured cells and mice. Mechanistically, N protein interacts directly with NLRP3 protein, promotes the binding of NLRP3 with ASC, and facilitates NLRP3 inflammasome assembly. More importantly, N protein aggravates lung injury, accelerates death in sepsis and acute inflammation mouse models, and promotes IL-1β and IL-6 activation in mice. Notably, N-induced lung injury and cytokine production are blocked by MCC950 (a specific inhibitor of NLRP3) and Ac-YVAD-cmk (an inhibitor of caspase-1). Therefore, this study reveals a distinct mechanism by which SARS-CoV-2 N protein promotes NLRP3 inflammasome activation and induces excessive inflammatory responses.

Many high quality studies have emerged from public databases, such as Surveillance, Epidemiology, and End Results (SEER), National Health and Nutrition Examination Survey (NHANES), The Cancer Genome Atlas (TCGA), and Medical Information Mart for Intensive Care (MIMIC); however, these data are often characterized by a high degree of dimensional heterogeneity, timeliness, scarcity, irregularity, and other characteristics, resulting in the value of these data not being fully utilized. Data-mining technology has been a frontier field in medical research, as it demonstrates excellent performance in evaluating patient risks and assisting clinical decision-making in building disease-prediction models. Therefore, data mining has unique advantages in clinical big-data research, especially in large-scale medical public databases. This article introduced the main medical public database and described the steps, tasks, and models of data mining in simple language. Additionally, we described data-mining methods along with their practical applications. The goal of this work was to aid clinical researchers in gaining a clear and intuitive understanding of the application of data-mining technology on clinical big-data in order to promote the production of research results that are beneficial to doctors and patients.

The glymphatic system is a glial-dependent waste clearance pathway in the brain, in place of lymphatic vessels, dedicated to drain away soluble waste proteins and metabolic products. Specifically, the glymphatic network serves as a "front end" for waste clearance, and is connected downstream to an authentic lymphatic network, associated with dura covering the brain as well as cranial nerves and large vessels at the skull exits. The anatomical and functional interconnections between these two networks are not completely understood. Several key physiological processes have been identified that control glymphatic transport function and waste clearance from brain. In this review, we aim to provide an overview and discussion of the concept behind the glymphatic system, current evidence, and controversies, while specifically focusing on the consequences of aging and evidence of its existence in human brain. Discovering novel strategies for optimizing and maintaining efficient brain waste clearance across the lifespan may in the future prove to be important for preventing cognitive decline and sustaining healthy aging.

OBJECT: Studies have suggested that depression was accompanied by oxidative stress dysregulation, including abnormal total antioxidant capacity (TAC), antioxidants, free radicals, oxidative damage and autoimmune response products. This meta-analysis aims to analyse the clinical data quantitatively by comparing the oxidative stress markers between depressed patients and healthy controls. METHODS: A search was conducted to collect the studies that measured the oxidative stress markers in depressed patients. Studies were searched in Embase, Medline, PsychINFO, Science direct, CBMDisc, CNKI and VIP from 1990 to May 2015. Data were subjected to meta-analysis by using a random effects model for examining the effect sizes of the results. Bias assessments, heterogeneity assessments and sensitivity analyses were also conducted. RESULTS: 115 articles met the inclusion criteria. Lower TAC was noted in acute episodes (AEs) of depressed patients (p<0.05). Antioxidants, including serum paraoxonase, uric acid, albumin, high-density lipoprotein cholesterol and zinc levels were lower than controls (p<0.05); the serum uric acid, albumin and vitamin C levels were increased after antidepressant therapy (p<0.05). Oxidative damage products, including red blood cell (RBC) malondialdehyde (MDA), serum MDA and 8-F2-isoprostanes levels were higher than controls (p<0.05). After antidepressant medication, RBC and serum MDA levels were decreased (p<0.05). Moreover, serum peroxide in free radicals levels were higher than controls (p<0.05). There were no differences between the depressed patients and controls for other oxidative stress markers. CONCLUSION: This meta-analysis supports the facts that the serum TAC, paraoxonase and antioxidant levels are lower, and the serum free radical and oxidative damage product levels are higher than controls in depressed patients. Meanwhile, the antioxidant levels are increased and the oxidative damage product levels are decreased after antidepressant medication. The pathophysiological relationships between oxidative stress and depression, and the potential benefits of antioxidant supplementation deserve further research.

As of March 24, 2020, novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for 379,661 infection cases with 16,428 deaths globally, and the number is still increasing rapidly. Herein, we present four critically ill patients with SARS-CoV-2 infection who received supportive care and convalescent plasma. Although all four patients (including a pregnant woman) recovered from SARS-CoV-2 infection eventually, randomized trials are needed to eliminate the effect of other treatments and investigate the safety and efficacy of convalescent plasma therapy. As of March 24, 2020, novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for 379,661 infection cases with 16,428 deaths globally, and the number is still increasing rapidly. Herein, we present four critically ill patients with SARS-CoV-2 infection who received supportive care and convalescent plasma. Although all four patients (including a pregnant woman) recovered from SARS-CoV-2 infection eventually, randomized trials are needed to eliminate the effect of other treatments and investigate the safety and efficacy of convalescent plasma therapy. An outbreak of novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection first appeared in Wuhan, China,1Guan WJ, Ni ZY, Hu Y, et al. Clinical characteristics of coronavirus disease 2019 in China [published online ahead of print February 28, 2020]. N Engl J Med. https://doi.org/10.1056/NEJMoa2002032.Google Scholar and rapidly spread to 171 countries. As of March 24, 2020, the virus has been responsible for 379,661 confirmed cases and 16,428 deaths worldwide. To date, no specific treatment has been recommended for SARS-CoV-2 infection except for meticulous supportive care.2Chen N. Zhou M. Dong X. et al.Epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in Wuhan, China: a descriptive study.Lancet. 2020; 395: 507-513Abstract Full Text Full Text PDF PubMed Scopus (13646) Google Scholar Numerous therapeutics have been explored or developed during the outbreak. A recent trial showed lopinavir-ritonavir has no treatment benefit for severe illness caused by SARS-CoV-2.3Cao B, Wang Y, Wen D, et al. A trial of lopinavir-ritonavir in adults hospitalized with severe Covid-19 [published online ahead of print March 18, 2020]. N Engl J Med. https://doi.org/10.1056/NEJMe2005477.Google Scholar Immunotherapy with virus-specific antibodies in convalescent plasma had been used as a last resort to improve the survival rate of patients with serious infectious diseases, such as severe acute respiratory syndrome, middle east respiratory syndrome coronavirus, Ebola virus disease, pandemic influenza A, and avian-origin influenza A.4Chen L, Xiong J, Bao L, et al. Convalescent plasma as a potential therapy for COVID-19 [published online ahead of print February 27, 2020]. Lancet Infect Dis. https://doi.org/10.1016/S1473-3099(20)30141-9.Google Scholar Previous reports have shown treatment with convalescent plasma collated from recovered patients could reduce the hospital stay and mortality of patients.5Soo Y.O. Cheng Y. Wong R. et al.Retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone treatment in SARS patients.Clin Microbiol Infect. 2004; 10: 676-678Abstract Full Text Full Text PDF PubMed Scopus (309) Google Scholar, 6Cheng Y. Wong R. Soo Y.O. et al.Use of convalescent plasma therapy in SARS patients in Hong Kong.Eur J Clin Microbiol Infect Dis. 2005; 24: 44-46Crossref PubMed Scopus (728) Google Scholar, 7Lai S.T. Treatment of severe acute respiratory syndrome.Eur J Clin Microbiol Infect Dis. 2005; 24: 583-591Crossref PubMed Scopus (92) Google Scholar However, the efficacy of convalescent plasma in critically ill patients with SARS-CoV-2 infection remains unclear. Herein, we report the disease course on four critically ill patients infected with SARS-CoV-2 and treated with supportive care and convalescent plasma. Figure 1 shows the clinical course of four critically ill patients infected with SARS-CoV-2. The first case is a 69-year-old woman with a history of hypertension who presented with fever for 2 days and clear sputum for 5 days. On January 30, the patient was admitted to Dongguan Ninth People’s Hospital because of positive reverse transcriptase polymerase chain reaction (RT-PCR) test of throat swab by Dongguan Center for Disease Control (CDC). A chest CT scan revealed bilateral ground-glass opacities primarily distributed along the pleura. Treatment with arbidol (200 mg three times daily), lopinavir-ritonavir (400 mg twice daily), interferon alpha inhalation (50 μg twice daily), and other supportive therapies was started. At 4 pm on February 4, the patient’s Po2 decreased to 56.5 mm Hg with an oxygenation index (OI) (Po2/Fio2) of 94 mm Hg. Significantly increased consolidation was observed in the right lung. The patient was transferred to the ICU of Dongguan People’s Hospital (a designated center for critical illness treatment) on February 5 and received invasive mechanical ventilation. Apart from antiviral drugs (lopinavir-ritonavir, oseltamivir, and interferon alpha), human albumin, zadaxin and immunoglobulin, and antibacterial and antifungal drugs were administrated because of coinfection with bacteria and Aspergillus. At 6:30 pm on February 11, the patient’s Po2 was 58 mm Hg. She experienced septic shock with BP of 89/44 mm Hg 5 h later. Hypohemoglobin (92 g/L) and bloody sputum under bronchoscopy suggested pneumorrhagia. A bedside chest radiograph showed obvious progression of disease. Although the patient was successfully rescued, follow-up chest radiographs showed continuous progression of pneumonia. A total of 900 mL O-compatible convalescent plasma was transfused to the patient in three batches; the first batch was given at 8 am on February 17 (200 mL), the second one was at 8 am on February 27 (400 mL), and the last one was at 8 am on February 28 (300 mL). The virus load of the patient on February 18 was 55 × 105 copies/mL, which significantly decreased to 3.9 × 104 copies/mL on February 28, and further decreased to 180 copies/mL on March 5. The patient was extubated and noninvasion ventilation was given on March 3. Chest CT scans obtained on February 27, March 6, and March 15 showed persistent absorption of consolidation. The results of two repeat RT-PCR tests of oropharyngeal swabs (with at least 1-day interval) performed on March 9 and 11 were negative. The patient was discharged on March 13. The second case was a 55-year-old man with a history of COPD who was admitted to a fever clinic of Xiangtan Central Hospital on February 5, 2020. He had nausea, poor appetite, and cough with clear sputum for 4 days. The results of RT-PCR assay of throat swab were positive for SARS-CoV-2 infection. A chest CT scan obtained on February 6 revealed interlobular septal thickening with honeycombing change in the right upper lung. The patient started to receive antiviral treatment, including arbidol (200 mg three times daily), lopinavir-ritonavir (500 mg twice daily), and interferon alpha-2b (5 million units twice daily). After 2 days, he complained of shortness of breath and his Po2 decreased to 50 mm Hg with an OI of 135 mm Hg. The patient was therefore diagnosed with ARDS and began to receive noninvasive mechanical ventilation and oxygen therapy through high-flow nasal cannula alternately. However, the conditions of the patient continued to deteriorate despite treatment with pulsed methylprednisolone. His Po2 oscillated between 46 and 83 mm Hg, and symptoms were not improved. Follow-up chest CT scans obtained on February 9 to 16 showed interstitial pneumonia extended to both lungs. At 3 pm on February 16, 200 mL convalescent plasma obtained from a patient recovered from SARS-CoV-2 infection in January 2020 was transfused to the patient. No adverse reactions were observed. One day later, his Po2 increased to 97 mm Hg with an OI of 198 mm Hg. All drugs were discontinued except for methylprednisolone. Chest images obtained on February 17 to 21 showed obvious absorption of interstitial pneumonia. Three repetitive RT-PCR test results were negative from February 20 to 22. The patient recovered and was discharged on February 23. He was asked to continue the quarantine at home for 14 days and receive home oxygen therapy. The third case was a 73-year-old man who was admitted to Dongguan Ninth People’s Hospital on February 2 because of self-reported dry cough for 4 days. He had a history of hypertension and chronic renal failure. On February 3, the patient was confirmed as being infected with SARS-CoV-2 by a virus RNA detection kit. At 11:30 pm, the patient developed acute respiratory failure with Po2 of 53 mm Hg and OI of 124 mm Hg; high-flow oxygen through face mask was given. He was then transferred to the isolation wards of the ICU of Dongguan People’s Hospital for further treatment. A chest radiograph showed bilateral infiltrative shadows. The viral load of the patient was as high as 85 × 105 copies/mL. The patient was treated with arbidol (200 mg three times daily), lopinavir-ritonavir (400 mg twice daily), oseltamivir (75 mg twice daily), and ribavirin and interferon alpha-2b (5 million units twice daily). On February 5, the patient was given tracheal intubation because of dyspnea and consistent decrease of oxygen saturation. On February 11, continuous renal replacement therapy (CRRT) was started on the patient. Laboratory tests obtained on February 14 showed significantly increased WBCs of 33.93 × 109/L and neutrophils of 31.08 × 109/L. He was diagnosed with multiple organ failure by clinical examination. On February 15, the patient developed septic shock and his BP decreased to 90/68 mm Hg with heart rate of 149 beat/min and respiratory rate of 30 breaths/min. A chest radiograph showed bilateral white lung. At 12:55 pm on February 15, the patient started to receive veno-venous extracorporeal membrane oxygenation, whereas the OI was unstable and symptoms were not improved. High-throughput DNA sequencing of sputum suggested Aspergillus infection. The patient was therefore treated with caspofungin and voriconazole. Eight transfusions of B-compatible convalescent plasma (2,400 mL) were given to the patient from February 16 to March 13. On February 21, the patient was confirmed positive for active pneumorrhagia, cystorrhagia, and GI bleeding. Antibody testing on February 27 indicated positive anti-SARS-CoV-2 IgG. The viral load was reduced (detailed values were not available). Follow-up chest radiographs showed absorbed infiltrative lesions but pneumothorax. Two repeat RT-PCR tests of sputum in deep lungs on March 16 and 17 (with at least 1-day interval) were negative and the serum IgM level decreased to the normal range. On March 22, the patient was transferred to the unfenced ICU for further treatment of underlying diseases and multiple organ failure. The fourth case was a 31-year-old pregnant woman (35 weeks and 2 days) who was admitted to Xiaolan People’s Hospital of Zhongshan on February 1 because of pharyngalgia for 4 days and fever (39.3°C) and difficulty breathing for half-day. The patient was confirmed as being infected with SARS-CoV-2 by Zhongshan CDC. A chest CT scan showed opacities in the lower lobe of the left lung. After admission, the patient developed severe ARDS, multiple organ dysfunction syndrome, and septic shock. Invasive ventilation and caesarean section were therefore given to the patient. Unfortunately, the newborn died of endouterine asphyxia. After the conditions turned stable, she was transferred to the Second People’s Hospital of Zhongshan (a designated hospital for SARS-CoV-2 treatment) at 1:04 am on February 2. Amounts of frothy sputum was observed under bronchofiberscope. Cardiac ultrasound suggested left ventricular enlargement with decreased systolic function. The patient received invasive ventilation and CRRT. Treatment with lopinavir-ritonavir (400 mg twice daily) and ribavirin (500 mg every 12 h) was started on February 2. Gram-positive bacteria were detected by blood culture, and imipenem and vancomycin were given to the patient. A chest radiograph showed increased consolidation and extended opacities. Oxygen saturation oscillated between 85% and 92% with an OI between 60 and 75 mm Hg. At 12 am on February 6, the patient started to receive veno-venous extracorporeal membrane oxygenation (flow rate: 3 L/min). Her OI was significantly improved (with a maximum of 200 mm Hg). Follow-up chest radiographs showed partial absorption of opacities. Left ventricular systolic function returned to normal. At 11:30 am on February 19, a 300-mL transfusion of convalescent plasma was given to the patient. On February 27, CRRT and extracorporeal membrane oxygenation (ECMO) were removed. On March 11, trachea cannula was removed and nasal oxygen was given to the patient. On March 6, 8, and 11, anti-SARS-CoV-2 IgM changed from positive to weakly positive to negative, whereas anti-SARS-CoV-2 IgG was persistently positive. Follow-up chest CT scan showed near-complete absorption of opacities. The results of two continual RT-PCR tests of BAL fluid on March 11 and 14 were both negative. The patient recovered from SARS-CoV-2 infection and was discharged on March 17. A recent retrospective review of 72,314 SARS-CoV-2-infected cases by the China CDC showed that 5% of cases were critical illness characterized by respiratory failure, septic shock, and/or multiple organ dysfunction or failure.8Zunyou W, McGoogan JM. Characteristics of and important lessons from the coronavirus disease 2019 (COVID-19) outbreak in China: summary of a report of 72 314 cases from the Chinese Center for Disease Control and Prevention [published online ahead of print February 24, 2020]. JAMA. https://doi.org/10.1001/jama.2020.2648.Google Scholar Around 48% of patients infected with SARS-CoV-2 had comorbid conditions, commonly cardiovascular diseases and diabetes.9Zhou F, Yu T, Du R, et al. Clinical course and risk factors for mortality of adult inpatients with COVID-19 in Wuhan, China: a retrospective cohort study [published online ahead of print March 11, 2020]. Lancet. https://doi.org/10.1016/S0140-6736(20)30566-3.Google Scholar Older adults with underlying diseases were more likely to have a higher Sequential Organ Failure Assessment score and higher risk of death. The treatment of SARS-CoV-2 infection faces compelling challenges. To date, no therapeutics have yet been proven effective for the treatment of the critical illness except for supportive care, including treatment with antiviral drugs, corticosteroids, immunoglobulins, and noninvasive or invasive mechanical ventilation. The most critically ill patients infected with SARS-CoV-2 have elevated levels of infection-related biomarkers and inflammatory cytokines, indicating potential bacterial coinfection caused by a dysregulated immune system.10Qin C, Zhou L, Hu Z, et al. Dysregulation of immune response in patients with COVID-19 in Wuhan, China [published online ahead of print March 12, 2020]. Clin Infect Dis. https://doi.org/10.1093/cid/ciaa248.Google Scholar Antibacterial drugs are therefore given to these patients. Management of critical SARS-CoV-2 infection is not different from management of most viral pneumonia causing respiratory failure. The principal feature of patients with the critical illness is the development of ARDS. ECMO is recommended by World Health Organization interim guidelines to support eligible patients with ARDS, while the use of which is restricted to specialized centers globally and technology challenges.11Ramanathan K, Antognini D, Combes A, et al. Planning and provision of ECMO services for severe ARDS during the COVID-19 pandemic and other outbreaks of emerging infectious diseases [published online ahead of print March 20, 2020]. Lancet Respir Med. https://doi.org/10.1016/S2213-2600(20)30121-1.Google Scholar In this study, two patients were treated with ECMO, but the efficacy was mixed. Apart from ARDS, other life-threatening conditions including septic shock and multiple organ dysfunction or failure may occur in a substantial proportion of patients with SARS-CoV-2-related critical illness, the management of which is according to current evidence-based guidelines.12De Backer D. Dorman T. Surviving sepsis guidelines: a continuous move toward better care of patients with sepsis.JAMA. 2017; 317: 807-808Crossref PubMed Scopus (67) Google Scholar In China, if the current therapeutic strategies are not satisfactory for critically ill patients, physicians might turn to convalescent plasma transfusion based on the Pneumonitis Diagnosis and Treatment Program for SARS-CoV-2 infection (Trial Version 7). Convalescent plasma has been used as a last resort to improve the survival rate of patients with severe acute respiratory syndrome infection. Previous evidence has proven that convalescent plasma treatment can significantly reduce the relative risk of mortality of patients,13Hung I.F. To K.K. Lee C.K. et al.Convalescent plasma treatment reduced mortality in patients with severe pandemic influenza A (H1N1) 2009 virus infection.Clin Infect Dis. 2011; 52: 447-456Crossref PubMed Scopus (534) Google Scholar which may be because antibodies from convalescent plasma might suppress viremia. The level of SARS-CoV-2 neutralizing antibodies in donor plasma could be important for the effectiveness of intervention. However, the level of neutralizing antibodies in donor plasma before transfusion cannot be determined. In this study, three patients were tested for either virus load or antibodies IgM and IgG. In the first case, SARS-CoV-2 virus load after convalescent plasma transfusion significantly dropped (from 55 × 105 to 3.9 × 104 to 180 copies/mL). Among the four patients, the time from transfusion to negative RT-PCR test results ranged from 3 to 22 days. The third and fourth cases produced anti-SARS-CoV-2 IgG approximately 14 days after convalescent plasma transfusion. Patients who survive critical illness might mount higher antibody responses, which can persist for longer periods compared with those with nonsevere disease.14Chen J. Zhu H. Horby P.W. et al.Specificity, kinetics and longevity of antibody responses to avian influenza A (H7N9) virus infection in humans.J Infect. 2020; 80: 310-319Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar The antibody levels, however, are confounded by other treatments, such as antiviral drugs, steroids, and IV immunoglobulin.15Luke T.C. Kilbane E.M. Jackson J.L. Hoffman S.L. Meta-analysis: convalescent blood products for Spanish influenza pneumonia: a future H5N1 treatment?.Ann Intern Med. 2006; 145: 599-609Crossref PubMed Scopus (487) Google Scholar A recent animal model indicated that antibodies produced from SARS-CoV-2 infection could protect from subsequent exposures.16Bao L, Deng W, Gao H, et al. Reinfection could not occur in SARS-CoV-2 infected rhesus macaques [published online ahead of print March 13, 2020]. bioRxiv. https://doi.org/10.1101/2020.03.13.990226.Google Scholar Our results indicate convalescent plasma might be a potential therapy for critically ill patients infected with SARS-CoV-2. We observed no serious adverse reactions associated with the transfusion of convalescent plasma. However, the relative contributions of supportive care, investigational therapies, and patient’s immune response on survival could not be determined. Whether convalescent plasma and/or supportive care provide any clinical benefit is unknown. The safety and efficacy of convalescent plasma transfusion in patients infected with SARS-CoV-2 should be studied within the context of a well-designed clinical trial.