Establishment and validation of an enzyme-linked immunosorbent assay for IgG antibody against cytomegalovirus based on pp150 antigen.

The development of HCMV vaccines for the prevention of congenital HCMV has been identified as a top priority by the Institute of Medicine (USA), and virus infection is an important endpoint for the efficacy evaluation of the candidate vaccines. However, it is technically difficult to capture the HCMV viremia or uremia in infected individuals because the viremia or uremia can be detected only transiently during acute infection, and most people who are infected are asymptomatic. Thus, it is much desired to develop a serological assay for effectively distinguishing anti-HCMV antibodies as a result of natural infection from those elicited by subunit antigen vaccination. In this study, five HCMV proteins other than antigens commonly used as subunit vaccine candidates were expressed in Escherichia coli and purified, and out of them, the HCMV tegument protein pp150 exhibited the most robust reactivity to seropositive sera and the faintest cross reaction to seronegative sera. With a coefficient of variability less than 15%, an ELISA based on pp150 antigen (pp150-ELISA) showed 100% (366/366) sensitivity and 100% (77/77) specificity (using neutralizing activity as a gold standard) and good quantitative correlation with an in-house ELISA based on virus lysate antigens. Taken together, these results indicate that pp150-ELISA, which has comparable performance to generally used assays based on virus lysate antigens, might provide a simple method to detect HCMV infection or reactivation and could be useful in future HCMV subunit vaccine efficacy trials.

Exon 35 skipping of FBN1 caused by c.4336G>A mutation may lead to Marfan syndrome / 中华检验医学杂志

Objective:To investigate the genetic etiology of a Marfan syndrome pedigree, and the impact of c.4336G>A variant on the splicing process of FBN1 gene.Methods:The proband was admitted to the Department of Cardiovascular Surgery of Xijing Hospital due to thoracic aortic aneurysm and dissection in August 2019. Multiplex PCR and next generation sequencing technology were used to detect 15 genes associated with hereditary aortic diseases in the proband. Then the pathogenic sites were further verified by Sanger sequencing, and above examinations were also performed among the family members of the proband. The effect of the mutation on mRNA splicing was predicted by splicing prediction software. RNAs from peripheral blood cells of the proband and the healthy person were extracted, and the effect of the mutation on mRNA splicing was verified by reverse transcription PCR and Sanger sequencing. The pathogenicity was analyzed by the recommendations from the American College of Medical Genetics (ACMG).Results:The gene panel detected a missense mutation of FBN1 gene (c.4336G>A) in the proband. Sanger sequencing results were consistent with that of panel. Sanger sequencing results showed that 4 family members were carriers of the same variant, and 3 out of the 4 family members presented signs of thoracic aortic aneurysm and dissection. The dbscSNV_ada_score and dbscSNV_rf_score software predicted that this mutation would lead to the occurrence of abnormal splicing of mRNA. The skipping of exon 35 was verified in the subsequent examinations by reverse transcription PCR and Sanger sequencing. The variant was classified as"pathogenic"according to ACMG guideline.Conclusion:FBN1 c.4336G>A mutation can cause the skipping of exon 35, and this might be the genetic mechanistic of severe cardiovascular abnormalities observed in this Marfan syndrome pedigree.

Monitoring of blood tacrolimus concentration and lymphocyte subsets in the first uterus transplant recipient in China and its clinical significance / 临床检验杂志

Objective To investigate the changes of peripheral blood tacrolimus concentration and lymphocyte subsets in the uterus transplant recipient,and provide the evidence for monitoring the immune status after uterus transplantation.Methods The peripheral blood tacrolimus concentrations of the uterus transplant recipient during 1 year after transplantation were measured with the microparticle enzyme immunoassay (MEIA).Meanwhile,the whole blood cell counts and lymphocyte subsets were determined by the blood analyzer and flow cytometer,respectively.Results The blood tacrolimus concentrations of the uterus transplant recipient in the first month and second month after transplantation were (13.51 ± 3.92) ng/mL and (15.58 ± 1.19) ng/mL,respectively.The lymphocyte absolute counts were normal before transplantation.At the fifth day after transplantation,the counts of CD3 + T lymphocytes,CD4 + T lymphocytes,CD8 + T lymphocytes and NK cells and the ratio of CD4/CD8 were significantly decreased.One week after transplantation,the counts of CD4 + T lymphocytes were recovered to the normal range and maintained,but its recovery was slower than that of CD8 + T lymphocytes.The ratio of CD4/CD8 ranged from 0.4 to 0.8 during 10 days after transplantation,and increased and maintained between 0.8 and 1.1 after that.The counts of NK cells increased gradually from the 10th day after transplantation,but still did not recover to the level before transplantation even at the 20th day after transplantation.However,the counts and percentages of B lymphocytes did not decrease but increased at the fifth day after transplantation,and recovered to normal gradually from the 10th day after transplantation.There was no significant correlation between the CD3 + T lymphocyte count and blood tacrolimus concentration.Conclusion The dynamic changes of blood lymphocyte subsets and tacrolimus concentration exist in the uterus transplant recipient,which need to be further verified by a large amount of clinical data.

Effect of dexmedetomidine on the renal function and expression of tight junction protein ZO-1 and occludin in the kidney of rats with septicopyemia / 中国比较医学杂志

Objective To investigate the effect of dexmedetomidine on the renal function and the expression of tight junction protein ZO-1 and occluding in the kidney of rats with septicopyemia induced by lipopolysaccharide.Methods Thirty adult male SD rats were randomly divided into 3 groups ( n=10 each):control group( group C) , LPS group ( group M) , and dexmedetomidine group ( group Dex ) .In the groups Dex and LPS, the rats were infused with saline and lipopolysaccharide (5 mg/kg, i.v.) respectively.In the Dex group, after intravenous injection of lipopolysaccharide (5 mg/kg) , the rats were firstly infused the loaded dose of dexmedetomidine (7μg/kg) for 15 minutes, and then changed to 5 μg/kg· min for 30 minutes.Blood samples were collected at 24 h later for measuring TNF-α, IL-1β, Scr and BUN.The kidney tissues were examined by histopathology.The expression of ZO-1 and occludin was detected by Western blot. Results Compared with the group C, the kidney tissue of group M was extensively damaged with tubular dilatation and inflammation, while reduced in the group Dex.Compared with the group C, the expressions of Scr, BUN, IL-1βand TNF-αwere all enhanced in the groups M and Dex ( P <0.05 ) , while the inflammatory factors in the group Dex were significantly lower than those of the group M ( P <0.05 ) .The Results The Western blot analysis showed that the expressions of protein ZO-1 and occludin in the group Dex were significantly higher than those of the group M (P<0.05). Conclusions Dexmedetomidine can improve the renal function of rats with septicopyemia, inhibit the acute renal injury and inflammation, increase the expression of protein ZO-1, and exert certain protective effect on the kidneys.

Establishment of a rat model of sepsis induced by muramyl dipeptide after scald burn / 中华麻醉学杂志

Objective To establish a rat model of sepsis induced by muramyl dipeptide (MDP) after scald burn.Methods Fifty SPF male Sprague-Dawley rats,aged 2-3 months,weighing 200-250 g,were randomly divided into 3 groups:control group (group C,n =10),scald group (group S,n =10) and MDP group (n =30).The rats were subjected to a third-degree scald burn covering 20% of total body surface area in groups S and MDP.The rats were only exposed to 20 ℃ water in group C.MDP 5 mg/kg was injected via the femoral vein at 24 h after scald bum in group MDP.Arterial blood samples were collected at 1,6 and 24 h after MDP injection in group MDP,at 24 h after scald burn in group S,or at 24 h after exposure to 20 ℃ water in group C for blood gas analysis and for measurement of white blood cell (WBC) and platelet (Plt) counts,serum aminotransferase (ALT),aspartate transferase (AST),total bilirubin (TB),creatinine (Cr) and blood urea nitrogen (BUN) levels,creatine kinase isoenzyme-MB (CK-MB) activity,and plasma tumor necrosis factor-α (TNF-α),interferon-γ(IFN-γ),interleukin-6 (IL-6),IL-10 and high mobility group box 1 protein (HMGB-1) levels.The rats were sacrificed after collecting blood samples,and heart,liver,lung,and kidney specimens were obtained for microscopic examination of pathologic changes.The activity of myeloperoxidase (MPO) in lung tissues was measured.Another 90 male Sprague-Dawley rats were randomly divided into 3 groups and treated as the method previously described for record of the survival rate within 72 h.Results Compared with C group,the plasma IL-6,IL-10,IFN-γ and HMGB1 levels,WBC count,serum ALT,AST,and BUN levels and MPO activity were significantly increased,and the survival rate within 72 h was decreased in S group,and the plasma TNF-α,IL-6,IL-10,IFN-γand HMGB-1 levels,serum ALT,AST,TB,BUN,Cr and CK-MB levels,MPO activity,PaCO2 and BE value were significantly increased,and WBC and PLT counts,pH value,PaO2 and survival rate within 72 h were decreased in MDP group (P ＜ 0.05).Compared with S group,the plasma TNF-α,IL-6,IFN-γ and HMGB-1 levels,serum ALT,AST,TB,BUN,Cr and CK-MB levels,MPO activity,PaCO2 and BE value were significantly increased,and WBC and Plt counts,pH value,PaO2 and survival rate within 72 h were decreased in MDP group (P ＜ 0.05).The pathologic changes of heart,liver,lung and kidney were obvious in S and MDP groups and severer in MDP group.Conclusion After a third-degree 20% total body surface area scald burn,MDP induces excessive production of inflammatory cytokines accompanying with multiple organ damage ; thus the model of sepsis is successfully established after scald burn in rats.

Effects of penehyclidine hydrochloride pretreatment on expression of β-arrestin-1 during sepsis-induced acute lung injury in mice / 中华麻醉学杂志

Objective To investigate the effects of penehyclidine hydrochloride (PHCD) pretreatment on β-arrestin-1 expression during sepsis-induced acute lung injury in mice.Methods Thirty female Kunming mice,weighing 18-20 g,were randomly divided into 3 groups (n =10 each):sham operation group (S group),sepsis group (CLP group) and PHCD group.Sepsis was induced by cecal ligation and puncture (CLP).In PHCD group,PHCD 0.45 mg/kg was injected intraperitoneally 1 h before CLP.The equal volume of normal saline was given instead in groups S and CLP.The mice were sacrificed at 12 h after CLP,bronchoalveolar lavage fluid (BALF) was collected for measurement of the total protein concentration,and the lungs were removed for determination of wet/dry lung weight ratio and expression of myosin light chain kinase (MLCK),vascular endothelial cadherin (VE-cad-herin) and β-arrestin-1 in lung tissues.The pathological changes of the lung were scored.Results Compared with group S,the lung injury score,wet/dry lung weight ratio and total protein concentration in BALF were significantly increased,MLCK expression was up-regulated and VE-cadherin expression was down-regulated in groups CLP and PHCD,β-arrestin-1 expression was down-regulated in group CLP and β-arrestin-1 expression was up-regulated in group PHCD (P ＜ 0.05 or 0.01).The lung injury score,wet/dry lung weight ratio,total protein concentration in BALF,and MLCK expression were significantly lower,while the expression of VE-cadherin and β-arrestin-1 was higher in PHCD group than in CLP group (P ＜ 0.05 or 0.01).Conclusion PHCD pretreatment can ameliorate acute lung injury through up-regulating β-arrestin-1 expression and reducing microvascular permeability in septic mice.

Effects of penehyclidine hydrochloric pretreatment on expression of β-arrestin-2 in the lung tissue in sepsisInduced acute lung injury in mice / 中华麻醉学杂志

Objective To investigate the effects of penehyclidine hydrochloric(PHC)pretreatment on the expression of β-arrestin-2 in the lung tissue in sepsis-induced acute lung injury in mice.Methods Thirty female Ktmming mice,aged 6 weeks,weighing 18-20 g,were randomly divided into 3 groups(n =10 each):sham operation group(group S); sepsis group(group CLP)and penehyclidine hydrochloric pretreatment group(group PHC).Sepsis was induced by cecal ligation and puncture(CLP)in groups CLP and PHC.Penehyclidine hydrochloric 0.45 mg/kg was injected intraperitoneally at 1 h before CLP in group PHC.While the equal volume of normal saline was given instead of penehyclidine hydrochloric in groups S and CLP.At 12 h of CLP,the animals were sacrificed,and the lung tissues were removed for determination of MPO activity(by colorimetry),IL-6 content(by ELISA),β-arrestin-2 mRNA and protein expression(by RT-PCR and Western blot respectively).Blood samples and bronchoalveolar lavage fluid were collected to calculate pulmonary vascular permeability index(PV PI).Results Compared with group S,PVPI,IL-6 content and MPO activity were significantly increased,the expression of β-arrcstin-2 protein was significantly down-regulaled while the expression of β-arrestin-2 mRNA was up-regulated in group CLP,and PVPI,IL-6 content and MPO activity were significantly incrcased,the expression of β-arrestin-2 protein was significantly up-regulated,while the expression of β-arrestin-2 mRNA was down-regulated in group PHC(P ＜ 0.05).Compared with group CLP,PVPI,IL-6 content,and MPO activity were significantly decreased,the expression of β-arrestin-2 protein was significantly up-regulated,while the expression of β-arrestin-2 mRNA was dow n-regulated in group PHC(P ＜ 0.05).Conclusion PHC pretreatment can attenuate the lung injury induced by sepsis in mice through up-regulating the expression of β-arrestin-2 protein.

Triterpene compounds from Cirsium setosum / 中国中药杂志

To investigate chemical constituents contained in cytotoxic petroleum ether extractive fractions from ethanol extracts of Cirsium setosum. The constituents were separated and purified by a combination of various chromatographic methods including silica gel, Sephadex LH-20, and preparative HPLC. Structures of the isolates were elucidated by spectroscopic methods including 1D, 2D NMR and MS methods. The compound structures were also determined by reference to literature. Twelve compounds were separated from the petroleum ether fraction of ethanolic extract and elucidated as lupenyl acetate (1), lupeol (2), lupenone (3), beta-amyrin (4), psi-taraxasterol (5), psi-taraxasteryl acetate (6), taraxasteryl acetate (7), marsformoxide B (8), alpha-amyrenone (9), beta-amyrenone (10), taraxasterone (11) and psi-taraxasterone (12). Of them, compounds 3, 5, 7-12 were separated from this genus for the first time.

Clinical observation of levonorgestrel-releasing intrauterine system in treatment of endometriosis / 中国医师进修杂志

Objective To evaluate the efficacy of levonorgestrel-releasing intrauterine system(LNGIUS) in the treatment of endometriosis. Methods Fifty-three patients suffering from endometriosis were placed LNG-IUS in uterine cavity after menstruation. The efficacy 3,6,12 months after LNG-IUS placement was observed and compared. Results The scores of dysmenorrhea, deep dyspareunia, menstrual anus pain 3,6,12 months after LNG-IUS placement were lower than those before LNG-IUS placement (P ＜ 0.05 ).CA125 [(21.8±12.5) kU/L]and destradiol [(78.8±45.5)ng/L] 12 months after LNG-IUS placement were improved than those before LNG-IUS placement and 3,6 months after LNG-IUS placement [CA 125:(36.3±17.5), (33.5±15.6), (28.2 ± 11.7) kU/L,destradiol: (40.4 ± 28.6), (42.5 ± 31.5), (56.5 ± 36.2) ng/L](P ＜ 0.05 ). The menstrual blood volume was less 6,12 months after LNG-IUS placement than that before LNG-IUS placement (P＜0.05), and the quality of life was improved( P ＜ 0.05 ). Conclusion LNG-IUS can be safe and effective treatment of patients with endometriosis, and less side effects.

Steroids from Monascus purpureus metabolite / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of Monascus purpureus metabolite.</p><p><b>METHOD</b>The compounds were isolated by column chromatography methods, and their structures were determined by spectroscopic methods.</p><p><b>RESULT</b>Eight compounds were isolated from the petroleum ether fraction of ethanolic extract and elucidated as stigmast-4-en-3-one (1), 3-oxo-24-methylenecycloarane (2), stigmasterol (3), 7beta-hydroxystigmasterol (4), 3beta-hydroxystigmast-5-en-7-one (5), 3beta-hydroxystigmast-5,22-dien-7-one (6), 5alpha, 8alpha-epidioxyergosta-6,22-dien-3beta-ol (7), sitosterol (8).</p><p><b>CONCLUSION</b>All of the compounds were isolated from this genu for the first time except compound 3 and 7.</p>