Expression of Ietalurus punetaus β-defensin based on recombinant Pichia pastoris / 生物工程学报

β-defensin is a primary protein immune factor in channel catfish's (Ietalurus punetaus) resistance to pathogenic microorganisms. Its primary structure contains a signal peptide composed of 24 amino acid residues at the N-terminal and a mature peptide composed of 43 amino acid residues at the C-terminal. The mature peptide region is responsible for the biological activity of β-defensin. In the present study, a recombinant strain of Pichia pastoris that produces channel catfish β-defensin, was constructed to realize the biosynthesis of channel catfish β-defensin based on eukaryotic expression. First, the β-defensin gene "IPBD" was isolated from the skin of channel catfish by RT-PCR. After linking it with the expression vector pPICZA, pPICZA-IPBD was transferred into competent P. pastoris X-33 cells to obtain recombinant P. pastoris strains. The yeast transformants with multi-copy gene inserts were obtained by using the culture medium containing 1 000 μg/mL zeocin. Using BMM culture medium (without amino nitrogen culture medium) instead of BMMY culture medium (with amino nitrogen culture medium), the fermentation and culture conditions of the recombinant strain were optimized, and the optimal conditions for producing channel catfish β-defensin were determined as follows: the expression was induced for 96 h with 1.0% methanol at 28 °C , 250 r/min. Purified protein with molecular weight of 5.98 kDa was obtained by nickel affinity chromatography, and MALDI-TOF/TOF mass spectrometry proved that it was the expected recombinant IPBD. The antibacterial test results showed that the inhibitory rates of recombinant IPBD on Gram-positive Staphylococcus aureus and Listeria monocytogenes and Gram-negative Pseudomonas aeruginosa were 69.6%, 71.6% and 65.8%, respectively. This study provides a recombinant DNA technique for the development of small molecule natural antibacterial peptide from fish.

Effect of icariin on early βdefensin2 and T cell subsets in rats after tracheotomy / 中南大学学报(医学版)

OBJECTIVES@#To investigate the effect of icariin (ICA) on early β-defensin-2 and T cell subsets in rats after tracheotomy.@*METHODS@#A total of 54 SPF male Sprague-Dawley rats were randomly divided into a normal control group (group A), a model group (group B), and a model+ICA treatment group (group C), with 18 rats in each group. A tracheotomy intubation model of the B and C group was prepared. After 6 h of surgery, ICA intervention was given to group C. Groups A and B were given the same amount of normal saline. Lung tissue, alveolar lavage fluid and peripheral blood were taken at 24 h, 72 h and 168 h, respectively. The expression of rat β-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of β-defensin-2 in alveolar lavage fluid and peripheral blood serum was detected by ELISA. The content of peripheral blood T cell subsets (CD3, CD4, CD8) was detected by flow cytometry, and the ratio of CD4/CD8 was calculated.@*RESULTS@#After tracheotomy, the levels of β-defensin-2 mRNA and β-defensin-2 in lung tissue from the group B were increased significantly at 24 h, then they were decreased gradually, and decreased most significantly at 168 h (0.05). The level of CD3 T cells in peripheral blood was significantly lower than that in the group A (0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum β-defensin-2 content, and peripheral blood CD3 and CD4 T cell levels were gradually increased, significantly higher than those in the group B (<0.05). CD8 T cell level was significantly lower than that in the group A at 24 h (<0.05), the CD4/CD8 ratio was significantly higher at 168 h than those in the group A or B (both <0.01).@*CONCLUSIONS@#ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of β-defensin-2 in lung tissue and alveolar lavage fluid, concomitant with increases in CD3 and CD4 T cells and CD4/CD8 ratio in peripheral blood while reduction in CD8 cells.

Role of human βdefensin 2 on preventing oxidized low-density lipoprotein induced monocyte foaming / 中华传染病杂志

Objective To clarify the role of human β-defensin2 ( hBD2) on preventing oxidized low-density lipoprotein (OX-LDL) induced human leukemic monocyte (THP-1) foaming.Methods The monocyte foaming model was established using THP-1 cell induced by OX-LDL and the model was identified by oil red staining.The hBD2 was overexpressed on THP-1 cells by using lentivirus system and the effect of hBD 2 overexpression on THP-1 cell foaming induced by OX-LDL was detected.The levels of inflammatory factors including tumor necrosis factor (TNF)-α, interleukin (IL)-1βand IL-6 in cell supernatant of each group were detected by enzyme-linked immunosorbent assay ( ELISA).Differences between the groups were compared by using the t test.Results The gene transfection efficiency of the cells was close to 100%at 72 h after infection. The hBD2 protein levels were 0.122 ±0.024 in the control group, 0.123 ±0.022 in Lv-control infection group and 0.981 ±0.183 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-3.175, P＝0.007).The relative levels of hBD2 mRNA at 72 h after virus infection were 0.131 ±0.021 in control group, 0.128 ±0.022 in Lv-control group and 1.001 ±0.105 in Lv-hBD2 infection group; and the level in control group was statistically higher than that in hBD-2 infection group (t＝-7.213, P＝0.003).The results of oil red staining showed that OX-LDL inducing THP-1 cells for 72 h could significantly induce lipid accumulation in cells.Overexpression of hBD2 could effectively inhibit lipid accumulation in THP-1 cells induced by OX-LDL.The expression of hBD2 mRNA in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝3.237, P＝0.004); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-6.021, P＝0.003).The level of hBD2 protein in THP-1 group was significantly higher than that in THP-1+OX-LDL group (t＝0.314, P＝0.006); and the difference was also significant when comparing THP-1+Lv-hBD2+OX-LDL group with THP-1+OX-LDL group (t＝-4.061,P＝0.007).The levels of TNF-α, IL-1βand IL-6 in the supernatant of THP-1 cells induced by OX-LDL for 72 h were significantly increased compared with those in THP-1group (t＝-3.825,-2.017 and -3.551, respectively; P＝0.007, 0.004 and 0.005, respectively). The levels of TNF-α, IL-1βand IL-6 in THP-1+Lv-hBD2+OX-LDL group were significantly lower than those in THP-1+OX-LDL group ( t＝4.132, 3.681, and 2.991, respectively; P＝0.003, 0.002, and 0.007, respectively).Conclusions hBD2 can effectively inhibit THP-1 foaming induced by OX-LDL, which may be related to its inhibition of inflammatory response.

Clinical Study on Pidotimod in the Treatment of Bronchial Asthma Complicated with Recurrent Respiratory Tract Infection in Children / 中国药房

OBJECTIVE:To investigate the clinical effects of pidotimod in the bronchial asthma complicated with recurrent re-spiratory tract infection,and its effects on immunoglobulin and related indexes. METHODS:A total of 120 bronchial asthma pa-tients with recurrent respiratory tract infection selected from our hospital during Mar. 2011-May 2013 were divided into trial group and control group according to random number table,with 60 cases in each group. Control group received routine corticosteroid therapy,and trial group was additionally given Pidotimod oral solution 0.4 g,po,bid,for 14 d,on the basis of control group. Clinical indexes(the times of respiratory infection,the duration of fever,cough,wheezing attack and antibiotics use),serum in-dexes [β-defensin-1(hBD-1),immunoglobulin A(IgA),IgG,IgM,UREA,ALT],the results of pharynx test before and after treatment,and the occurrence of ADR were observed in 2 groups. RESULTS:Before treatment,there was no statistical signifi-cance in clinical indexes,serum indexes,the results of pharynx test between 2 groups(P>0.05). After treatment,clinical indexes of trial group were significantly lower or shorter then before treatment or control group,while serum levels of hBD-1,IgA and IgG were significantly higher than before treatment or control group,with statistical significance(P0.05). The types and number of pathogenic bacteria of respiratory tract infection were decreased significantly in 2 groups,and the trial group was significantly less then the control group,with statistical significance(P<0.05). No obvious ADR was found in 2 groups. CONCLUSIONS:Pidotimod shows good clinical effects on bronchial asthma complicated with recurrent re-spiratory tract infection,can improve immunity and reduce the types and number of pathogenic bacteria with good safety.

Antifungal effects of synthetic human β-defensin 3-C15 peptide

OBJECTIVES: The purpose of this ex vivo study was to compare the antifungal activity of a synthetic peptide consisting of 15 amino acids at the C-terminus of human β-defensin 3 (HBD3-C15) with calcium hydroxide (CH) and Nystatin (Nys) against Candida albicans (C. albicans) biofilm. MATERIALS AND METHODS: C. albicans were grown on cover glass bottom dishes or human dentin disks for 48 hr, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and 300 µg/mL), CH (100 µg/mL), and Nys (20 µg/mL) for 7 days at 37℃. On cover glass, live and dead cells in the biomass were measured by the FilmTracer Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin, normal, diminished and ruptured cells were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to a two-tailed t-test, a one way analysis variance and a post hoc test at a significance level of p = 0.05. RESULTS: C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (≥ 100 µg/mL). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (≥ 100 µg/mL) showed significant fungicidal activity compared to CH group (p < 0.05). CONCLUSIONS: Synthetic HBD3-C15 peptide (≥ 100 µg/mL) and Nys exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm.

Effect ofβ-defensin 2 gene silencing on the inflammatory response induced by Haemophilus influenzae type B combined vaccine immunization in rats / 中华传染病杂志

Objective To investigate the effect of β‐defensin 2 (rBD‐2 ) silencing on the inflammatory response induced by Haemophilus influenzae type B vaccine immunization in rats .Methods A total of 144 SPF Sprague Dawley (SD) rats were divided into four groups .Group 1 were immunized with Haemophilus influenzae type B vaccine .Group 2 were immunized with Haemophilus influenzae type B vaccine and administered with lentivirus Lv‐shRNA‐rBD‐2 by intratracheal instillation .Group 3 were administered with the lentivirus Lv‐shRNA‐rBD‐2 by intratracheal instillation .Group 4 were blank controls .Each group was set with 12 rats . The serum was collected at 12 ,24 and 72 hours after immunization ,and tumor necrosis factor (TNF)‐α,interleukine (IL)‐1β and IL‐10 were assessed by enzyme‐linked immuno sorbent assay (ELISA ) .Expression levels of rBD‐2 in lung tissues of rats were measured by western blotting . Results The recombinant virus was successfully produced by a lipofectmaine transfection method .At 12 ,24 ,and 72 h of immunization ,serum levels of TNF‐αof rats in group 1 were (82 .0 ± 8 .0) ,(155 .0 ± 18 .2) ,and (272 .0 ± 32 .5) pg/mL ,respectively ,which were all higher than those in group 4 ([55 .0 ± 6 .2] ,[52 .0 ± 5 .8] ,and [56 .0 ± 4 .8] pg/mL ,respectively ;t=16 .034 ,P=0 .043 ;t=12 .411 ,P=0 .035 ;t=10 .530 ,P=0 .018 ,respectively) .Serum levels of TNF‐αat 12 ,24 ,and 72 h of immunization in group 2 were (66 .0 ± 8 .2) ,(90 .0 ± 12 .6) ,and (108 .0 ± 13 .6) pg/mL ,respectively ,which were all significantly lower than group 1 (t=12 .115 ,P=0 .039 ;t=12 .830 , P=0 .033 ;t=15 .522 ,P=0 .012 ,respectively) .At each time point ,serum levels of IL‐1βand IL‐10 in group 1 were all significantly higher than those in group 4 (IL‐1β:t=18 .032 , P=0 .048 ;t=15 .824 , P=0 .039 ;t=13 .518 , P=0 .021 ,respectively ;IL‐10 :t=15 .410 , P=0 .045 ;t=14 .294 , P=0 .032 ;t=13 .375 ,P=0 .013 ,respectively) .Serum levels of IL‐1βand IL‐10 at each time points in group 2 were all significantly lower than those in group 1 (IL‐1β:t=19 .012 , P=0 .043;t=16 .991 , P=0 .034;t=14 .862 , P=0 .027 ,respectively ;IL‐10 :t=15 .134 , P=0 .048 ;t=15 .264 , P=0 .036 ;t=11 .408 , P=0 .024 ,respectively) .Seventy‐two hours after immunization of SD rats ,the relative content of rBD‐2 protein in lung tissue of rats significantly increased .Protein level in group 1 was significantly higher than that in group 4 (t=10 .582 ,P=0 .035) ,while protein level in group 2 was significantly lower than that in group 1 (t=13 .250 ,P=0 .027) .Conclusions rBD‐2 gene silencing can improve the inflammatory response induced by Haemophilus inf luenzae type B combined vaccine in rats.

Effects of recombinant fusion protein interleukin-17F/His on expression of immune-related inflammatory factors in mouse model with Streptococcus pneumoniae infection / 中华实用儿科临床杂志

Objective To evaluate the effects of recombinant fusion protein interleukin-17F/His (IL-17F/His) on expression of immune-related inflammatory factors in murine models with Streptococcus pneumoniae (SP) infection.Methods BALB/c mice were randomly divided into SP infection group,fusion protein intervention group and the normal control group.Before intranasal infection with SP,the mice were treated with PBS or IL-17F/His respectively.The number of bacteria and leukocytes in bronchoalveolar lavage fluid (BALF) was counted,and the mRNA levels of β-defensin-2 (Defb2),macrophage inflammatory protein (MIP)-lα and MIP-2β in lung tissue were detected by real-time fluorescent quantitative PCR,the concentrations of MIP-1 α,MIP-2β,IFN-γ and IL-4 in BALF,supematants of spleen cell and mediastinal lymph node cell were assayed by enzyme linked immunosorbent assay.The expressions of IL-17F and Defb2 in lung tissues were observed with immunocytochemistry.Results 1.Compared with the SP infection group,in the fusion protein intervention group,the total number of WBC (209.00 ± 18.23) was higher,the number of neutrophil,macrophage and lymphocyte[(8.67 ±2.16) × 106/L,(193.50 ± 16.50) × 106/L,(6.83 ± 1.17) × 106/L] were higher in BALF(U/F =38.097,28.854,32.942,27.810,all P ＜ 0.05),while the number of bacteria [2.75 (1.15-3.09) × 103/L] was significantly lower(P =0.02).2.Compared with SP infection group,the expression levels of Defb2 mRNA and MIP-1α mRNA(31.64 ±4.97,5.81 ± 1.09) in lung tissues were higher in the fusion protein intervention group,while the expression level of MIP-2β mRNA (6.69 ± 2.05) was lower (t =4.889,2.306,3.536,all P ＜ 0.05).3.Compared with the SP infection group,the concentration of MIP-2β [(64.15 ± 13.41) ng/L] was significantly decreased and concentration of IL-4 [(92.28 ± 4.52) ng/L] was significantly increased in BALF in the fusion protein intervention group(H/F =159,289,40.767,all P ＜0.01).4.Compared with the SP infection group,the concentrations of MIP-2β and IL-4 [(255.02 ± 13.95) ng/L,(107.02 ± 7.53) ng/L] were both significantly increased in spleen cell culture supematants in the fusion protein intervention group,the concentrations of MIP-1 α and IFN-γ [(413.61 ± 24.23) ng/L,(98.88 ± 5.84) ng/L] were both significantly decreased (all P ＜ 0.05).5.Immunocytochemistry analysis revealed that the expressions of IL-17F and Defb2 were both higher in the fusion protein intervention group than those in the SP infection group; there was no expression in the normal control group.Conclusions Intranasal inoculation of recombinant fusion protein IL-17F/His can promote pulmonary neutrophil and macrophage recruitment and increase bacterium clearance,and enhance the host defense against SP infection through increa-sing the expression of defensins,regulating the levels of MIP,IL-4 and IFN-γ.

Research progress and prospect of human β-defensin 3 and its derivatives in ophthalmology / 中华实验眼科杂志

Currently,antimicrobial resistance has become an urgent global public health problem.Human defensin,a family of innate immune peptides,plays an important role in the control of infectious disease because of its low resistance to microbiological infection.However,so far there is seldom report on industrial production of bioactive defensin.Human β-defensin 3 (HBD3) is a member of human innate antimicrobial peptides.It has broad-spectrum antimicrobial activity and function in a variety of inflammation diseases.In addition,HBD3 is considered as a good candidate for antibiotics,because it has a strong killing activity to antibiotics-resistant microbes and resistance to highsalt environment,it is especially valuable for the treatment of ocular surface inflammatory diseases.This review covers the in vitro research on the production of HBD3,optimization of structure-activity and its antimicrobial potential for ocular surface infection,which may provide a supportive evidence for the commercial production of HBD3 and other antimicrobial peptides in biopharmaceutical area.

Expressions of human β defensin-2 in eutopic and ectopic endometrium of women with adenomyosis / 中国综合临床

Objective To investigate the expression of human β defensin-2(hβD-2)in the eutopic and ectopic endometrial tissue in patients with adenomyosis and in women with normal endometrium.Methods Twenty-five hysteromyoma patients with adenomyosis(AM) and 25 hysteromyoma patients without endometriosis (EMS) were selected and divided into three groups:AM ectopic endometrium group,AM eutopic endometrium group and control group( endometrial tissue in patients with hysteromyoma).The level of mRNA expressions of hβD-2,interleukin-1β ( IL-1β ),interleukin-6 ( IL-6 ) and tumor necrosis factor-α (TNF-α) was investigated quantitatively using Real Time PCR (RT-PCR) and the corresponding protein level was detected by immunohistochemistry.Results Comparing the expression of hβD-2,IL-1β,IL-6,TNF-α genes among the three groups,there was no significant difference between ectopic endometrium group and eutopic endometrium group and there was also no significant difference between eutopic endometrium group and the control group.( P ＞ 0.05 ).The expression of hβD-2 and IL-1β were 0.0320 (0.0095 ～ 0.0690 ) and 0.0427 ( 0.0038 ～ 0.0975 ) in the ectopic endometrium group,and they were 0.0034(0.0025 ～0.0424) and 0.0080(0.0040 ～0.0251 ) in the control group,respectively.They were both significantly higher in ectopic endometrium group than in the control group (P ＜ 0.05 ).In the ectopic endometrium group hβD-2 expression was positively correlated with the level of TNF-α ( r =0.857,P =0.014 ),and it had no correlation with both IL-1β and IL-6 ( r =0.750,P =0.052 ; r =0.464,P =0.464; respectively)Conclusion HβD-2 might not play an important role in the formation of adenomyosis.It may be related to no significant up-regulation of inflammatory factors in ectopic lesion tissue.

Anti-infection effect of human beta-defensin-2 gene therapy in a rat urinary tract infection model / 中华泌尿外科杂志

Objective To assess the human β-defensin-2 ( hBD2 ) gene therapeutic efficacy in a rat urinary tract infection (UTI) model via intravesical liposome-mediated gene transfer.Methods Fifty-six female Wistar rats (class SPF) weighting 1 80 -220 were randomly divided into an experiment group and a control group ( each n =28 ).The animals were administered either 250 μl recombinant pCAGG-hBD2 or control vector pCAGG intravesically.After 48 h,rats in both groups were infected via intravesical inoculation with 200 μl of the bacterial suspension of UTI89 ( 1 × 108 CFU/ml).The rats were sacrificed at 4,24,48,and 72 h post-inoculation.The bladders were aseptically removed and bisected.One half was fixed in neutral buffered formalin for histological analysis.The remaining half-bladders were homogenized and titered for surviving bacteria.The clean-catch urine sample from each rat was collected in sterile before they were killed for bacterial titers determined and WBC counted.Results Numbers of bacterial colony-forming unit in urine and bladders from hBD2 gene treated UTI rats were significantly lower than those from the control vector administered UTI rats at 24,36,and 72 h after infection ( P ＜ 0.05 ).The amount of WBC in urine was significantly less in the defensin group than in the control group.In addition,in vivo expression of hBD2 could reduce mucosa damage,interstitium edema and inflammatory cells infiltration in UTI animals.Conclusions The successful inhibition of UTI progression could be obtained with hBD2 gene therapy.Recombinant beta-defensin-2 could kill UTI89 in vivo and suppress the subsequent infection-induced inflammatory responses.

Study on the mechanism of human-β-defensin-2 expression in human vaginal epithelial cells induced by Lactobacillus cell wall extract / 中华微生物学和免疫学杂志

Objective To investigate the molecular and cell signal transduction mechanisms by Lactobacillus cell wall extract(LCWE)inducing human-β-defensin-2(hBD-2)expression in human vaginal epithelial cells.Methods The induction of hBD-2 in human vaginal epithelial cells(WZV-1)by LCWE was observed using real-time PCR and Western blot.After stimulating WZV-1.the activation of NF-κB and p38MAPK signaling pathways were determined by Western blot.The induction of hBD-2 in WZV-1 cells by LCWE was observed with signaling pathways inhibitors of NF-κB and p38MAPK using real-time PCR and Western blot.Results The results showed that LCWE significantly upregulated hBD-2 expression in the time and dose-dependent manner.The maximal stimulatory effect of LCWE on the expression of hBD-2mRNA in WZV-1 cells were observed at the concentration of 50μg/ml after treatment for 8 h.After stimulation by 50μg/ml LCWE,Western blot analysis demonstrated that the phosphorylation of p38MAPK increased at 0.5 h significantly,peaked at 1 h,moreover the concentration of NF-κB in nucleus increased at 0.5 h significantly(P<0.05),peaked at 2 h.Blocking with inhibitor of NF-κB and(or)p38MAPK pathways results in decreased levels of HBD-2 expression.Conclusion These findings suggest that p38MAPK and NF-κB pathways play the important roles in induction of hBD-2 expression by LCWE in human vagihal epithelial cells.

Expression and function of human β-defensin-2 in patients with peptic ulcer / 中国医师进修杂志

Objective To investigate the relationship between the pathophysiology of Helicobacter pylori induced antral gastritis and the expression of human β -defensin (HBD) -2 in peptic ulcer before and after Helicobacter pylori eradication.Methods Patients in peptic ulcer with Helicobacter pylori-posilive (23 cases) or Helicobacter pylori-negative (20 cases) were included.And 30 normal individuals were as controls.Biopsied gastric mucosa specimens from Helicobacter pylori-positive or Helicobacter pylori-negative individuals were done and normal controls were used.The specimens were examined for pathophysiology diagnosis and HBD-2 expression by immunohistochemistry before and after Helicobacter pylori eradication.Results Helicobacter pylori infection was correlated with the histological severities of both inflammation activity and atrophy in antral gastritis (r =0.574,0.640,P ＜ 0.01 ).The expression of HBD-2 was positively correlated with Helicobacter pylori infection in all the specimens (r =0.533,0.577,P ＜ 0.01 ).Immunohistochemistry using anti-HBD-2 antiserum revealed that HBD-2 expression and inflammation activity had significantly decreased in gastric specimens obtained after Helicobacter pylori eradication(P ＜ 0.01 ).The expression of HBD-2 had no variance in the case failing in Helicobacter pylori eradication (P ＞0.05).Conclusions Helicobacter pylori infection induces HBD-2 expression in the human gastric epithelium.HBD-2 inhibits the growth of Helicobacter pylori in vitro,which suggests that HBD-2 plays an antibacterial or inhibition role in Helicobacter pylori induced gastritis.In other words,the absence of HBD-2 should enhance the histological inflammation of Helicobacter pylori induced antral gastritis.