Changes of heparin-binding protein in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells and neutrophils / 中华烧伤杂志

Objective: To investigate the changes of heparin-binding protein (HBP) in severe burn patients during shock stage and its effects on human umbilical vein endothelial cells (HUVECs) and neutrophils in vitro. Methods: Prospective observational and experimental research methods were used. Twenty severe burn patients who met the inclusion criteria and were admitted to the Department of Burns and Plastic Surgery of Affiliated Suzhou Hospital of Nanjing Medical University from August to November 2020 were included in severe burn group (12 males and 8 females, aged 44.5 (31.0, 58.0) years). During the same period, 20 healthy volunteers with normal physical examination results in the unit's Physical Examination Center were recruited into healthy control group (13 males and 7 females, aged 39.5 (26.0, 53.0) years). Enzyme-linked immunosorbent assay (ELISA) method was used to detect the protein expression levels of HBP and tissue inhibitor of metalloproteinase 1 (TIMP-1) in plasma of patients within 48 hours after injury in severe burn group and in plasma of volunteers in healthy control group. The correlation between protein expression of HBP and that of TIMP-1 in the plasma in the two groups was analyzed by Pearson correlation analysis. The fourth passage of HUVECs in logarithmic growth phase were used for the experiment. The HUVECs were divided into normal control group with routine culture (the same treatment below) and recombinant HBP (rHBP)-treated 12 h group, rHBP-treated 24 h group, and rHBP-treated 48 h group with corresponding treatment according to the random number table (the same grouping method below), and the mRNA expression of TIMP-1 in cells was detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction. The HUVECs were divided into normal control group and rHBP-treated 48 h group with corresponding treatment, and the protein expression of TIMP-1 in the cells was detected by Western blotting. The HUVECs were divided into normal control group, rHBP alone group, aprotinin alone group, and rHBP+aprotinin group treated with the corresponding reagents (with the final molarity of rHBP being 200 nmol/L and the final concentration of aprotinin being 20 μg/mL, respectively), cultured for 48 h, and ELISA was used to detect the protein expression of TIMP-1 in the culture supernatant of cells. The neutrophils were isolated from the peripheral venous blood of the aforementioned 10 healthy volunteers by immunomagnetic bead sorting, and the cells were divided into normal control group, recombinant TIMP-1 (rTIMP-1) alone group, phorbol acetate (PMA) alone group, and rTIMP-1+PMA group treated with corresponding reagents (with the final concentration of rTIMP-1 being 500 ng/mL and the final molarity of PMA being 10 nmol/L, respectively). After being cultured for 1 h, the expression of CD63 protein in cells was detected by immunofluorescence method, the positive expression rate of CD63 protein in cells was detected by flow cytometry, and the protein expression levels of HBP and myeloperoxidase (MPO) in the culture supernatant of cells were detected by ELISA. The normal control group underwent the above-mentioned related tests at appropriate time points. The number of samples was 3 in each group of cell experiment. Data were statistically analyzed with chi-square test, Mann-Whitney U test, Kruskal-Wallis H test, and Tamhane's T2 test. Results: The protein expression levels of HBP and TIMP-1 in the plasma of patients in severe burn group were 404.9 (283.1, 653.2) and 262.1 (240.6, 317.4) ng/mL, respectively, which were both significantly higher than 61.6 (45.0, 68.9) and 81.0 (66.3, 90.0) ng/mL of volunteers in healthy control group (with Z values of -5.41 and -5.21, respectively, P<0.01). The correlation between the protein expression of HBP and that of TIMP-1 in the plasma of volunteers in healthy control group was not strong (P>0.05). The protein expression of HBP was significantly positively correlated with that of TIMP-1 in the plasma of patients in severe burn group (r=0.64, P<0.01). Compared with that in normal control group, the mRNA expression of TIMP-1 in HUVECs was significantly increased in rHBP-treated 12 h group, rHBP-treated 24 h group, and rHBP-treated 48 h group (with t values of -3.58, -2.25, and -1.26, respectively, P<0.05). Western blotting detection showed that compared with that in normal control group, the protein expression of TIMP-1 in HUVECs in rHBP-treated 48 h group was significantly enhanced. After 48 h of culture, compared with that in normal control group, the protein expression level of TIMP-1 in the culture supernatant of HUVECs in rHBP alone group was significantly increased (t=9.43, P<0.05), while the protein expression level of TIMP-1 in the culture supernatant of HUVECs didn't change significantly in aprotinin alone group or rHBP+aprotinin group (P>0.05); compared with that in rHBP alone group, the protein expression level of TIMP-1 in the culture supernatant of HUVECs in rHBP+aprotinin group was significantly decreased (t=4.76, P<0.01). After 1 h of culture, the trend of CD63 protein expression in neutrophils detected by immunofluorescence method and that by flow cytometry were consistent in each group. After 1 h of culture, compared with that in normal control group, the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells in rTIMP-1 alone group all had no significant changes (P>0.05), while the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells were all significantly increased in PMA alone group and rTIMP-1+PMA group (with t values of 2.41, 3.82, 5.73, 1.05, 4.16, and 1.08, respectively, P<0.05 or P<0.01); compared with that in PMA alone group, the positive expression rate of CD63 protein in the neutrophils and the protein expression levels of HBP and MPO in the culture supernatant of cells in rTIMP-1+PMA group were all significantly decreased (with t values of 5.26, 2.83, and 1.26, respectively, P<0.05 or P<0.01). Conclusions: The expression level of HBP in the plasma of severe burn patients is increased during shock stage. HBP can induce HUVECs to secrete TIMP-1 in vitro, and TIMP-1 can reduce the expression of CD63 molecule in human neutrophils.

Re-understanding the physiological and pathophysiological roles of neutrophils / 中华烧伤杂志

Neutrophils have always been considered as a short-lived and homogeneous cell type in the innate immune system, which have limited pro-inflammatory or anti-inflammatory effects. However, in recent 10 years, the understanding of neutrophils has been undergoing some kind of revival as researches progressed. The researches on the heterogeneity of neutrophils and the mechanism of their interaction with other immune cells have promoted the researchers to re-understand the physiological and pathophysiological roles of neutrophils. In the following decades, with the development of single-cell sequencing technology, spatial transcriptome sequencing technology, and multi-omics combined sequencing technology, researchers will have a better understanding of the biological behaviors of neutrophils. This paper briefly reviews the biological behaviors of neutrophils and their roles in various diseases in recent years.

Retrospective Analysis of Hematological Phenotypes in Patients with Gene Mutation and Deletion α-Thalassemia / 中国实验血液学杂志

OBJECTIVE@#To explore the differences between hematological phenotypes of patients with different genotypes in gene mutations and deletion α- thalassemia.@*METHODS@#By screening the α- thalassemia gene test results in the First Affiliated Hospital, Sun Yat-Sen University from January 2015 to April 2020, the patients with mutation and deletion α- thalassemia were obtained, then the differences between hematological phenotypes of patients with different genotypes were analyzed.@*RESULTS@#There were 96 patients with mutation combined with deletion α- thalassemia from the results of 24 054 α- thalassemia patients screened out, including 79 patients with non-deletion Hb H disease (α@*CONCLUSION@#The hematological phenotype changes caused by α

Growth and Adhesion to Cells of Candida albicans Labeled by Calcofluor White and Fluorescent Concanavalin A / 中山大学学报(医学科学版)

@#【Abstract】 【Objective】To assess effect of calcofluor white and fluorescent dye labeled concanavalin A in Candida albicans growth and adhesion of cells.【Methods】Yeast cells of 20 strains of Candida albicans were labeled by calcofluor white and Alexa fluor 488 conjugated concanavalin A respectively. The growth of labeled Candida albicans were tested by counts of colony forming unit. Then yeast cells of Candida albicans were co-cultured with macrophages and enterocytes for half an hour. The adhesion of labeled Candida albicans to macrophages and enterocytes were observed by fluorescence microscopy.【Results】No difference was observed on the number of colony forming units between calcofluor white-labeled groupand control group(P=0.942). Also,no difference was observed on the number of colony forming units between Alexa fluor 488 conjugated concanavalin A-labeled group and control group. However,either the number of calcofluor white-labeled Candida albicans or Alexa fluor 488 conjugated concanavalin A-labeled Candida albicans that bound to macrophages was less than that in control group(P=0.000,respectively). Either the number of calcofluor white-labeled Candida albicans or Alexa fluor 488 conjugated concanavalin A-labeled Candida albicans that bound to enterocytes was less than that in control group(P = 0.000,respectively). Hyphae were observed in control group but not in calcofluor white group after yeast cells of Candida albicans were co-cultured with cells for half an hour.【Conclusions】Growth of Candida albicans was not changed,while its adhesion to cells was reduced after its labeling by calcofluor white and Alexa fluor 488 conjugated concanavalin A.The germination of Candida albicans was affected when it had been labeled by calcofluor white.

A study on the association among serum insulin,IGFBP3, and endometrial cancer risk in Chinese women / 预防医学

Objective Toexploretheassociationamongseruminsulin,IGFBP3,andendometrialcancerriskinChinese women.Methods SeruminsulinandIGFBP3weredetectedbyELISAmethodin206patientswithendometrialcarcinoma and 310 healthy women.Using logistic regression analysis after adjustments for BMI,serum glucose and triglycerides to exploretheassociationamongthetwoindicatorsandtheriskofendometrialcarcinoma.Results Increasedinsulinwere found in the women with endometrial carcinomas as compared with that of controls [Mean ±SD:insulin (14.84 ±16.72) uU·mL-1 in women with cancer versus (8.13 ±9.40)uU·mL-1 in controls,P<0.01].However,serum IGFBP3 was not significantly higher in women with endometrial cancer [Mean ±SD:IGFBP3 (1.76 ±2.44)mg·L-1 in women with cancer versus (1.57 ±1.80)mg·L-1in controls,P>0.05].The risk for endometrial cancer was significantly higher in the upper quartile relevant to the lowest quartile of serum insulin,and lower in the upper quartile of serum IGFBP3 (P<0.05).Logistic regression analysis showed that serum insulin was the risk factor of endometrial carcinoma(OR=2.34, 95%CI:1.32 -4.14),after adjusting obesity/overweight status,serum glucose,total cholesterol,total glyceride,and HDL-C.Conclusion HyperinsulinemiawasanindependentriskfactorforendometrialcarcinomasinChinesewomen. However,the protective role of increased serum IGFBP3 should be validated further.