Preliminary evaluation of the safety and efficacy of oral human antimicrobial peptide LL-37 in the treatment of patients of COVID-19, a small-scale, single-arm, exploratory safety study

Background& AimsThe Coronavirus Disease 2019 (COVID-19) has become a global epidemic and has caused a lasting and huge loss of life security, economic development and social stability in more than 180 countries around the world. Unfortunately, there is still no specific treatment for COVID-19 till now, therefore, at this point, all potential therapies need to be critically considered. LL-37 is one of the best-studied human antimicrobial peptide (AMPs) that has a broad-spectrum activity against bacteria and viruses. The use of living, genetically modified organisms (GMOs) is an effective approach for delivery of therapeutic proteins. The aim of this study was to determine the safety and efficacy of the Lactococcus lactis which has been genetically modified to produce the therapeutic human antimicrobial peptide LL-37 (herein after referred to cas001) in the patients of COVID-19. MethodsFirstly we constructed genetically modified food-grade probiotic, Lactococcus lactis, with sequence of seven tandem repeats of mature human LL-37 under control of the nisin-inducible nisA promoter to produce the cas001. A total of 20 healthy SD rats, half male and half female (There were five male and five female in the control group, the same in treatment group) were used to observe the acute toxic reaction and death after daily administration of cas001 for three weeks, which helps to provide necessary reference basis for clinical dose selection, verificaition of toxic reaction and possible target organs. According to the estimated clinical dosage of 1 x 108CFU /kg/day, considering the conversion of body surface area, the dose for rats should be multiplied by 6.17 to 6 x 108 CFU/kg/day. We administrated 100 times higher dose at 6 x 1010 CFU/ /kg/day to rats. In order to investigate the pharmacokinetics of cas001, male SD rats (body weight 250-300g, 1 x 1010 /animal, n=3) were given oral administration of LL-37 bacteria powder. The concentration of LL-37 in the blood before and after gavage was detected by ELISA kit (Hycult biotechnology Cat# HK321). Human clinical study was approved by Ethics committee of Chinese PLA General Hospital (S2020-074-04) and a total of 11 patients with mild symptoms were enrolled in Wuhan hankou hospital and Huoshenshan hospital. They were enrolled voluntarily and all patients signed informed consent. Among them, there were 5 males and 6 females, aged 55 {+/-} 12 (36-70) years old, and the duration from onset to medication enrollment was 35 {+/-} 19 (5-68) days. 6 patients were nucleic acid positive and 5 patients were nucleic acid negative when they were enrolled. All patients received the oral drug cas001 treatment according to requirement(1 x 109 CFU/capsule, 3 capsules/time, three times a day for 3weeks), with an average follow-up time of 33 {+/-} 15 days (see table 1 for the results). O_TBL View this table: org.highwire.dtl.DTLVardef@1845d29org.highwire.dtl.DTLVardef@1004b38org.highwire.dtl.DTLVardef@4a741aorg.highwire.dtl.DTLVardef@c73d5org.highwire.dtl.DTLVardef@188b563_HPS_FORMAT_FIGEXP M_TBL O_FLOATNOTable 1.C_FLOATNO O_TABLECAPTIONSerum biochemical detection in rats two weeks after gavage Serum biochemistry detection of rats at the end of two weeks (Vehicle[male] v.s. LL-37[male], n=5; Vehicle[female] v.s. LL-37[female], n=5). *represent p value<0.05. C_TABLECAPTION C_TBL FindingsWestern blot analysis shows that reasonable amount of LL-37 were induced by different concentrations of nisin, which means we have successfully constructed cas001. In the pre-clinical safety evaluation test, after three weeks administration of cas001, no adverse effects were observed on the rats body weight, food and water intake, hematological or serum biochemical parameters. The results showed that the LD50 of cas001 was higher than that of the 100 times of the expected clinical dose of 6 x 1010 CFU/day. These results showed that cas001 could be safe in animal experiments. In addition, rat pharmacokinetics results showed that the serum concentration of LL-37 reached peak 2 hours after gavage of cas001 and returned to basal level 6 hours after gavage. During study period, the volunteers did not feel any discomfort while taking the cas001 capsules, and two hours after oral administration, the concentration of LL-37 were increased in healthy volunteers. cas001 shows definite effect in the improvement of gastrointestinal symptoms and is possible to have effects in improving the systemic symptoms and respiratory symptoms and may play a role in the improvement of results of nucleic acid test and lung CT test. 11 patients enrolled showed good compliance, tolerance, subjective feeling and actively interacted with the doctors. None of the patients had any adverse reactions. ConclusionsBased on above observations, we conclude here that as an oral anti-viral agent, cas001 displayed good safety profiles. It is very hard to reach conclusion of clinical outcomes related to the cas001, although changes of several symptoms indicate encouraging findings.

Detecting 41 mineral elements in human whole blood by inductively coupled plasma-mass spectrometry / 中国职业医学

OBJECTIVE: To establish a method for detecting 41 mineral elements such as beryllium, magnesium and calcium in whole blood by inductively coupled plasma-mass spectrometry(ICP-MS). METHODS: After the whole blood samples were diluted 1∶20 in a mixed solution containing 0.10% Triton X-100 and 0.50% nitric acid, 41 mineral elements in whole blood samples such as beryllium, magnesium and calcium were determined by the ICP-MS, using scandium, rhodium and iridium as the internal standard elements. RESULTS: The linear correlation coefficient was >0.999 0 in these 41 elements such as beryllium, magnesium and calcium in the whole blood. The detection limit was 0.006-247.000 μg/L, the quantification lower limit was 0.020-822.000 μg/L. The recovery rate of standard addition was 84.00%-109.40%. The within-run and between-run relative standard deviation were 0.66%-7.34% and 0.56%-8.22%, respectively. The samples could be stored at-20 ℃ for at least 14 days. CONCLUSION: This method is simple, rapid and sensitive, with good precision and reliable results. It can be used for detecting multiple mineral elements in whole blood of normal and occupational population.

Correlations Between Single Nucleotide Polymorphisms, Cognitive Dysfunction, and Postmortem Brain Pathology in Alzheimer's Disease Among Han Chinese / 神经科学通报·英文版

In this study, the distribution of five Alzheimer's disease (AD)-related single nucleotide polymorphisms (SNPs) in the Han population was examined in combination with the evaluation of clinical cognition and brain pathological analysis. The associations among SNPs, clinical daily cognitive states, and postmortem neuropathological changes were analyzed in 110 human brains from the Chinese Academy of Medical Sciences/Peking Union Medical College (CAMS/PUMC) Human Brain Bank. APOE ε4 (OR = 4.482, P = 0.004), the RS2305421 GG genotype (adjusted OR = 4.397, P = 0.015), and the RS10498633 GT genotype (adjusted OR = 2.375, P = 0.028) were associated with a higher score on the ABC (Aβ plaque score, Braak NFT stage, and CERAD neuritic plaque score) dementia scale. These results advance our understanding of the pathogenesis of AD, the relationship between pathological diagnosis and clinical diagnosis, and the SNPs in the Han population for future research.

Detecting 12 elements in human biological samples by ICP-MS method / 中国职业医学

OBJECTIVE: To establish a method for detecting the contents of 12 toxic elements such as beryllium,vanadium and thallium in human whole blood,urine and hair by inductively coupled plasma-mass spectrometry( ICP-MS).METHODS: Whole blood( 0.25 mL) and urine( 10.00 mL) were diluted 20 times into suspension by 0.1% TritonX-100 + 0.5% nitric acid,and analyzed by ICP-MS.The hair( 0.20 g) was mixed with nitric acid and digested by microwave,then made up to 10.00 mL with purified water.The analysis was performed by ICP-MS.RESULTS: The linear correlation coefficients of all the 12 elements,such as beryllium,vanadium,chromium,manganese,cobalt,nickel,arsenic,cadmium,tin,antimony,tellurium and lead were≥0.999 5.The detection limit for whole blood and urine was 0.097-1.995 μg/L,and the detection limit for hair was 0.001-0.012 μg/g.The recovery rates for whole blood,urine and hair were 92.3%-105.0%,93.7%-115.5% and 92.5%-111.0%,respectively.The within-run relative standard deviation( RSD) were 0.7%-5.8%,0.8%-4.6% and 2.2%-8.4%,respectively; the between-run RSD were 1.6%-7.1%,3.5%-7.5% and 2.8%-8.8%,respectively.CONCLUSION: This method has good accuracy,high sensitivity and good precision,which is suitable for rapid screening for various elements in human biological samples.

Clinical analysis of sudden cardiac death in aged patients / 中国基层医药

Objective To study the clinical features of aged patients with sudden cardiac death(SCD) for improving the resuscitation rate. Methods Hie usual etiology of SCD,electrocardiogram(ECG),echocardiography, dynamic electrocardiography(DCG), X-ray and electrolytes were analyzed. Results Most sudden death had inducement. Electrolytes disorder,ventricular ectopic beats,high grade atrioventricular block, auxocardia, left ventricular hypertrophy, cardiac insufficiency and myocardial ischemia could cause serious arrhythmias (.all P <0.05). Conclusion It could be related to heart functional class,left ventricular enlargement,myocardial ischemia,hypokalemia in aged patients with SCD.