Research progress on the development of human neutrophil elastase inhibitors / 药学学报

Human neutrophil elastase (hNE) is a serine proteolytic enzyme mainly distributed in neutrophils. When the balance between anti-hNE protein and hNE is broken, excessive release of hNE can cause the occurrence of various diseases. Therefore, inhibition of hNE is a promising therapeutic strategy. In this paper, the structure, action mechanism, physiological function of hNE and the development of hNE inhibitors were briefly summarized, in order to provide information for the related research.

Research Progress of Human Neutrophil Elastase Inhibitors and Their Application in Diverse Diseases / 中国药学杂志

To comprehensively understand the research status of human neutrophil elastase inhibitors (HNEI) and their clinical application prospect, the papers about novel HNEI discovered since 2011 and diseases related to human neutrophil elastase (HNE) in addition to pulmonary diseases were summarized. The results showed that a lot of highly selective and potent HNE inhibitors have been discovered since 2011. HNE participates in the development of many diseases. In addition to the infectious and inflammatory pulmonary diseases reported in the past, it is also associated with ischemia-reperfusion injury, rheumatoid arthritis, autoimmune diabetes, nephritis, cancer and other diseases. The development of novel HNEI with high potency and low toxicity has been an important direction for HNE-related diseases.

Utility of procalcitonin, human neutrophil lipocalin and neutrophil CD64 in early diagnosis of bacterial infection / 中国感染与化疗杂志

Objective To examine the expression levels of procalcitonin (PCT),neutrophils apolipoprotein (human neutrophil lipocalin,HNL) and neutrophil CD64 (CD64) in the blood of patients with bacterial infection and investigate their utility in early diagnosis and treatment of bacterial infection.Methods A total of 210 patients with confirmed infection who were treated in hospital from February 2013 to May 2017 were enrolled.The patients were classified into bacterial infection group (105 cases) and viral infection group (105 cases).Additionally,a cohort of 80 healthy subjects were randomly selected from health checkup during the same period as the control group.PCT and HNL were determined on the UPT up-converting phosphor microbial immunity analyzer provided by Beijing Hotgen Biotech Company.BD FACS calibur flow cytometer was used to measure and calculate CD64 percentage.Results The levels of PCT,HNL,CD64 and WBC were compared between bacterial infection group,viral infection group and control group.The between-group difference was statistically significant (P＜0.05).The CD64 and WBC levels were significantly different between viral infection group and control group (P＜0.05),but the PCT and HNL levels were not different significantly between viral infection group and control group (P＞0.05).The area under the ROC curve of PCT,namely AUC PCT,was 0.855,and AUC (HNL) was 0.930,AUC (CD64) 0.928,and AUC (WBC) 0.729.The cutoff value of PCT,HNL and CD64 for diagnosis of bacterial infections was ＞0.79 ng/mL,＞87.43 ng/mL,and ＞9.01％,respectively.Conclusions Bacterial infection is associated with elevated levels of PCT,HNL and CD64,which can be used in early diagnosis of bacterial infections.HNL may provide the highest diagnostic value.

Gene Frequency of Human Neutrophil Alloantigen-3 in the Korean Population / 대한수혈학회지

BACKGROUND: In the Korean population, gene frequencies of Human Neutrophil Alloantigen-1 (HNA-1), HNA-2, HNA-4, and HNA-5 were determined using serological and genotyping methods. However, no study assessing the gene frequencies of HNA-3 among the Korean population has been conducted. The aims of this study were to report the gene frequencies of HNA-3, to estimate the risk of HNA-3a (or HNA-3b) alloimmunization, and to secure donors of granulocytes for anti-HNA-3 antibodies among the Korean population. METHODS: Genotyping of HNA-3a and HNA-3b genes of 110 healthy and unrelated Korean donors was performed using a polymerase chain reaction with sequence-specific primers. RESULTS: The observed frequencies of HNA-3a and HNA-3b were 0.695 and 0.305, respectively, among the Korean population. The HNA-3a gene frequency of Koreans was significantly lower than those of American Whites and Germans (P<0.05). The risk of HNA-3a and HNA-3b alloimmunization due to pregnancy (transfusion) was 0.065 (0.084) and 0.147 (0.250), respectively, among the Korean population. The risk of HNA-3a alloimmunization was significantly higher in the Korean population than in the German and American White populations (P<0.05). CONCLUSION: The gene frequencies of HNA-3 and the risk of HNA-3a (HNA-3b) alloimmunization due to pregnancy or transfusion among the Korean population were determined. We also identified individuals who were HNA-3a/3a or HNA-3b/3b for the granulocyte panel which could be used for anti-HNA antibody identification.

Effects of tumor necrosis factor-α converting enzyme on mucous hypersecretion in inflammatory airway / 中南大学学报(医学版)

Objective: To investigate the effect of tumor necrosis factor-α converting enzyme (TACE) on mucous hypersecretion in inlf ammatory airway. Methods: Mucous hypersecretion model of human lung adenocarcinoma cells A549 was induced by human neutrophil elastase (HNE), and TNF-α converting enzyme inhibitor-1 (TAPI-1), an inhibitor of TACE, was chosen for the inference study. The expression of MUC5AC and TACE was examined. hT e cells were divided into 5 groups: a negative control group, HNE1 (15 nmol/L) group, HNE2 (25 nmol/L) group, HNE3 (50 nmol/L) group and TAPI-1 group. RT-PCR was used to examine MUC5AC and TACE mRNA expression. The protein expression of TACE and MUC5AC was examined by Western blot and ELISA, respectively. Results: HNE induced the TACE and MUC5AC mRNA and protein expression in a dose-dependent manner. Compared with the control group, the increases were all signiifcantly increased in the three dosages of HNE group (P<0.01). The HNE-induced TACE and MUC5AC mRNA and protein expression were dramatically attenuated in the presence of TAPI-1, an inhibitor of TACE (P<0.01). Conclusion: TACE participated cell signalling pathway of airway mucous hypersecretion, and could down regulation the level of inlfammation airway mucous hypersecretion.

Clinical value of NHL detection in the differential diagnosis of bacterial and viral infections of elderly patients with acute respiratory infection / 国际检验医学杂志

Objective Toinvestigatetheclinicalvalueofhumanneutrophillipocalin(HNL)detectioninthedifferentialdiagnosis of bacterial and viral infections of elderly patients with acute respiratory infection .Methods 142 elderly patients with respiratory infection were divided the bacteria group (96 cases) and the virus group (46 cases) according to their infections ,42 healthy people in the corresponding period were enrolled as the control group .Enzyme-linked immunosorbent assay and highly sensitive dry chemi-cal particles enhanced immune turbidity assay were employed to detect their blood HNL and C-reactive protein(CRP) ,respectively , and virus-specific antibodies detection were performed simultaneously .Results Compared the blood HNL ,CRP levels and their positive rates of patients in bacteria group with those in the virus group ,control group ,respectively ,differences showed statistically significant(P0 .05) .Antibiotic treatment before and 24 ,48 and 72 hours after ,the concentrations of HNL were (216 .8 ± 64 .1) , (192 .0 ± 41 .2) ,(158 .0 ± 54 .5) and (87 .0 ± 12 .4)μg/L ,respectively ,while those of CRP were (50 .9 ± 40 .9) ,(46 .2 ± 18 .3) , (39 .6 ± 9 .6) and (12 .6 ± 9 .8) mg/L ,respectively .Sensitivity ,specificity ,positive predictive value and negative predictive value of HNL detection were 90 .6% ,90 .9% ,91 .5% and 89 .9% ,respectively ,which were higher than those of CRP (88 .5% ,85 .2% , 86 .7% and 87 .2% ,respectively) ,with statistically significant difference(P<0 .05) .Conclusion NHL detection possesses impor-tant significance in differential diagnosis between bacterial and viral infections of elderly patients with acute respiratory infection .

Intracellular expression of Dectin-1 mediates the killing of Candida albicans by human neutrophils in a manner dependent on ROS production / 中华微生物学和免疫学杂志

Objective To investigate the mechanism hy which Dectin-1 mediates the killing of Candida albicans(C.albicans) by human neutrophils in a manner dependent on the production of reactive oxygen species (ROS).Methods After stimulation with FITC-C.albicans at a multiplicity of infection (MOI) of 10 for 30 or 60 min,PE-anti-human Dectin-1 monoclonal antibody (2.5 μg/106 cells) was used to detect the expression of Dectin-1 in human neutrophils by flow cytometry.For Dectin-1 inhibition test and ROS assay,human neutrophils (2×106/ml) were respectively pre-incubated with different concentrations of blocking antibody (0.5,1,2.5 and 5 μg/ml) for 60 min at 4℃,and then with 25 μmol/L 2′,7′-Dichlorofluorescein diacetate for another 20 min at room temperature.Afterwards,under stimulation with live C.albicans at a MOI of 10,the rate of intracellular ROS production over time in blocking and control groups was measured continuously at 10 min intervals for up to 120 min.In addition,localization of Dectin-1 and ROS in human neutrophils was observed by confocal/two-photon laser scanning microscopy after stimulation with live C.albicans.For the detection of candicidal activity,after pre-treatment with different concentrations of Dectin-1 blocking antibody as mentioned above,neutrophils were stimulated with live C.albicans (MOI=1) for 60 min,serial dilutions of cell lysate were plated onto yeast agar,and CFU were enumerated after incubation at 37℃ for 48 h.The candicidal activity was represented as [1-(CFUblocking group/CFUbuffer)] × 100％.Results Under stimulation with FITC-C.albicans at a MOI of 10 for 30 and 60 min,positive percentage of intracellular Dectin-1-expressing neutrophils increased significantly when compared with initial level (0 min,8.32％ ; 30 min,16.82％ ; 60 min,23.88％) (versus 0 min,P＜0.01).However,positive percentage of cell-surface Dectin-1-expressing neutrophils remained almost unchanged after stimulation for 30 and 60 min (versus 0 min,P＞0.05).Upon blockage of Dectin-1,the stimulated ROS generation (R2=0.306,P＜0.01) and candicidal activity (R2=0.251,P＜0.01) of neutrophils were partly and irreversibly inhibited in a dose-dependent manner when compared with control group.In addition,the intracellular Dectin-1 is recruited and co-distributed with ROS on the surface of phagocytized C.albicans as observed by confocal microscopy.Conclusion Taken together,these results demonstrated that an internalized expression pattern of human Dectin-1 might contribute to the ROS-dependent killing of serum-opsonzied C.albicans which was phagocytized by human neutrophils.

Effects of Scutellarin on MUC5AC Mucin Production Induced by Human Neutrophil Elastase or Interleukin 13 on Airway Epithelial Cells

Scutellarin is a flavonoid extracted from a traditional Chinese herb, Erigeron breviscapus. The present study investigated the effect of scutellarin on MUC5AC mucin production and the possible mechanism. Human bronchial epithelial 16 (HBE16) cells were pretreated with scutellarin for 60 min, and then exposed to human neutrophil elastase (HNE) or interleukin (IL)-13 for 12 hr. RT-PCR and ELISA were performed to measure the amount of MUC5AC mucin production. The results showed that scutellarin inhibited MUC5AC expression both in mRNA and protein level induced by HNE in a concentration-dependent manner. However, scutellarin failed to inhibit MUC5AC mucin production induced by IL-13. To investigate the intracellular mechanisms associated with the effect of scutellarin on MUC5AC mucin production, western blotting was carried out to examine the phosphorylation of protein kinase C (PKC), signal transducer and activator of transcription 6 (STAT6) and extracellular signal-regulated kinase 1/2 (ERK1/2). The phosphorylation of PKC and ERK1/2 was attenuated after treatment with scutellarin, whereas STAT6 was not significantly affected. Therefore, it is suggested that scutellarin down-regulates MUC5AC mucin production on HBE16 cells via ERK-dependent and PKC-dependent pathways.

NF-kappaB and CREB Are Involved in IL-8 Production of Human Neutrophils Induced by Trichomonas vaginalis-Derived Secretory Products

Trichomonas vaginalis is a flagellated lumen-dwelling extracellular protozoan parasite that causes human trichomoniasis via sexual intercourse. Human neutrophils play a crucial role in acute tissue inflammatory responses in T. vaginalis infection. In this study, we investigated the signaling mechanism of neutrophil responses when stimulated with T. vaginalis-derived secretory products (TvSP), which were collected from 1x10(7) live trichomonads. Incubation of human neutrophils isolated from peripheral blood with TvSP induced up-regulation of IL-8 protein secretion. In addition, stimulation with TvSP induced phosphorylation of NF-kappaB and CREB in neutrophils. Moreover, TvSP-induced IL-8 production was also significantly inhibited by pretreatment of neutrophils with ikappaB inhibitor or CREB inhibitor. These results suggest that transcription factors NF-kappaB and CREB are involved in IL-8 production in human neutrophils induced by stimulation with T. vaginalis infection.

Roles of theaflavins in inflammatory airway mucus hypersecretion / 上海第二医科大学学报

Objective To investigate the roles of theaflavins(TFs) in airway mucus hypersecretion induced by human neutrophil elastase(HNE). Methods Human lung adenocarcinoma cell A549 was stimulated by HNE for mucus hypersecretion,and TF monomers(TF1,TF2 and TF3) were used for intervention.The effects of TF monomers on viability of A549 cells were examined by MTT method.After the effective doses of TFs were determined,A549 cells were divided into 4 groups for experiment.In control group,A549 cells were cultured with serum-free medium.In HNE treatment group,A549 cells were treated with HNE(50 nmol/L) for 24 h.In TF monomer intervention groups,A549 cells were pre-treated with TF1,TF2 or TF3(50,100 or 200 ?g/mL) for 24 h,and were then treated with HNE for another 24 h.In AG1478 intervention group,A549 cells were pre-treated with AG1478(5 ?mol/L),an epidermal growth factor receptor blocker for 30 min,and were then treated with HNE for another 24 h.The changes in mucin(MUC) after treatment by different doses of TF1,TF2 and TF3,and by TF3(200 ?g/mL) for different time(12 h,24 h and 36 h) were detected.The changes in MUC5AC mRNA expression and MUC5AC protein expression were detected by RT-PCR and ELISA,respectively. Results The MUC5AC mRNA expression and protein expression in HNE treatment group were significantly higher than those in control group(P

Effect of CD14, Toll-like receptors, cytoskeletal inhibitors and NF-kappaB inhibitor on MMP-8 release from human neutrophils induced by E. coli lipopolysaccharides / 대한치주과학회지

OBJECTIVE: MMP-8 is a neutrophil enzyme and its level increases in some inflammatory diseases, including periodontal disease. We knew that the lipopolysaccharide of E.coli(E-LPS) induced MMP-8 release from human neutrophils. E-LPS is known to induce the production and release of inflammatory cytokines through CD14, Toll-like receptor(TLR). In the present study, we investigated whether MMP-8 release by E-LPS is induced via CD14-TLR pathway and the cellular mechanism of MMP-8 release in human neutrophils. MATERIAL AND METHODS: Human neutrophils were isolated from the peripheral blood of healthy donors and pre-incubated in medium containing antibodies against CD14, anti-TLR2 and anti-TLR4 or several inhibitors of microtubules and microfilaments and then incubated with E-LPS. The cells were treated TPCK and E-LPS simultaneously. The MMP-8amount in the culture medium was determined using ELISA. RESULTS: E-LPS increased MMP-8release from neutrophils and its induction was inhibited by anti- CD14 and anti-TLR4 but not by anti-TLR2 antibodies. The inhibitors of micro- tubule and microfilament polymerization significantly decreased E-LPS-induced MMP- 8release. TPCK inhibited E-LPS-induced MMP-8 release. CONCLUSION: These results suggest that MMP-8 release is induced by E-LPS via the CD14-TLR4 signal pathway in human neutrophils and may be depedent on microtubule and microfilament systems and NF-kappaB pathway.

HNA frequencies in the Kinh ethnic of Vietnam population, determined by polymerase chain reaction with sequence-specific primers

The human neutrophil antigens (HNA) are often targets of granulocyte antibodies causing immune neutropenia. Typing for HNA is useful in a veriety of clinical situations. The author developed a method for genotyping for HNA-1 by means of the PCR ampification with sequence specific primer (PCR-SSP) technique. The typing results were available within 5 hours each time for one blood sample tested. In 61 granulocytes samples, the gen frequencies were 64.7% for HNA-1a and 35.2% for HNA-1b. The HNA-1a gene is more frequent in the Kinh population than the HNA-1b

Western blot of the expression of J chain-human netrophil peptide-1 / 医学研究生学报

Objective:Defensins from human neutrophils exhibit a broad antimicrobial activity.The transfection of the gene fused by human neutrophil peptide 1(HNP1) with the J chain into other cells may induce the in vitro expression of HNP1 to play antimicrobial roles.The authors analyzed the influencing factors on the expressed proteins of HNP1 and the J chain gene in COS-7 cells.Methods: Western blot was used to analyze the technical factors influencing the HNP1 protein expression.Results: Recombinant J-HNP1 was expressed in the cells successfully.Conclusion: The expression of J-HNP1 can be detected inside and outside the cells by Western blot.

The Relationship between Human Neutrophil Elastase and Coronary Arterial Dilatation in Kawasaki Disease

PURPOSE: Kawasaki disease is notorious for coronary arterial complication which is usually developed as a febrile disease in early childhood. Increased polymorphonucleus(PMN) cell levels in acute phases may be associated with the pathophysiology of Kawasaki disease. We studied the relationship between coronary arterial dilatation and elastase activity which was excreted from PMN cell and roles as an important factor for vasculitis. METHODS: Ten patients diagnosed with Kawasaki disease in Yonsei University Medical Center were examined between November, 2001 and January, 2002. In addition, 15 patients with other febrile diseases were also examined. Echocardiography was done in patients with Kawasaki disease on the first day of admission and four weeks after the onset of the disease. At each time, venous samples were drawn and separated into plasma and leukocytes. In patients with other febrile disease, samples were drawn on admission. Elastase activities in plasma and neutrophil extracts were measured. RESULTS: The significant increased plasma elastase activity, 6.19+/-0.74 U/mL, found in Kawasaki disease patients compared with the other febrile disease patients, 4.86+/-1.17 U/mL(P<0.05). And there was no significance between the above two diseases in terms of the elastase activity in neutrophil extracts. The relationship between initial elastase activity and the coronary arterial complication which was shown in subacute phase wasn't significant. CONCLUSION: Plasma elastase activity was increased in Kawasaki disease significantly, but the initial plasma elastase activity in the acute phase could not reflect the range of coronary arterial complication.

Construction and expression of eukaryotic expression vector harboring human neutrophil peptide 1 / 解放军医学杂志

Objective To explore the possibility of human neutrophil peptide 1 (HNP1) gene engineering, we construct the eukaryotic expression vector carrying HNP1 gene. Methods With RNA extracted from human neutrophil cell as template, cDNA encoding mature HNP1 was amplified by RT-PCR, and then it was inserted into vector pMD18-T. After restriction endonuclease digestion and DNA sequencing confirmation the gene was subcloned into eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO to construct a recombinant expression plasmid pcDNA3.1/V5-His-TOPO/HNP1, then the recombinant plasmid was transfected into COS-7 cells by lipofectamine, and the expressed product was identified by biotin-avidin enzyme-linked immunosorbent assay ( BA-ELISA). Results The sequence of HNP1 completely matched those published in GenBank, thus eukaryotic expression vector pcDNA3.1/V5-His-TOPO/HNP1 was constructed correctly. The ELISA results showed that the eukaryotic expression plasmid pcDNA3.1/V5-His-TOPO/HNP1 could temporarily express HNP1 in COS-7 cells. Conclusion The successful construction and expression of pcDNA3.1/V5-His-TOPO/HNP1 pave the way for the stable expression HNP1 in mammalian engineering cells.

Molecular characteristics of the inhibition of human neutrophil elastase by nonsteroidal antiinflammatory drugs

Nonsteroidal antiinflammatory drugs(NSAIDs) are known as clinically effective agents for treatment of inflammatory diseases. Inhibition of cyclooxygenase has been thought to be a major facet of the pharmacological mechanism of NSAIDs. However, it is difficult to ascribe the antiinflammatory effects of NSAIDs solely to the inhibition of prostaglandin synthesis. Human neutrophil elastase (HNElastase; HNE, EC 3.4.21.37) has been known as a causative factor in inflammatory diseases. To investigate the specific relationship between HNElastase inhibition and specificity of molecular structure of several NSAIDs, HNElastase was purified by Ultrogel AcA54 gel filtration, CM-Sephadex ion exchange, and HPLC (with TSK 250 column) chromatography. HNElastase was inhibited by aspirin and salicylate in a competitive manner and by naproxen, ketoprofen, phenylbutazone, and oxyphenbutazone in a partial competative manner, but not by ibuprofen and tolmetin. HNElastase-phenylbutazone-complex showed strong Raman shifts at 200, 440, 1124, 1194, 1384, 1506, and 1768 cm(-1). The Raman bands 1194, 1384, and 1768 cm(-1) may represent evidences of the conformational change at -N=N-phi radical, pyrazol ring, and -C=O radical of the elastase-drug complex, respectively. Phenylbutazone might be bound to HNElastase by ionic and hydrophobic interaction, and masked the active site. Inhibition of HNElastase could be another mechanism of action of NSAIDs besides cyclooxygenase inhibition in the treatment of inflammatory diseases. Different inhibition characteristics of HNE-lastase by NSAIDs such as aspirin, phenylbutazone-like drugs and ineffective drugs could be important points for drawing the criteria for appropriate drugs in clinical application.

Solid-phase enzyme. linked immunosorbent assay and its clinical significance for human neutrophil elastase / 中国病理生理杂志

The role of neutrophil in the pathogenesis of many diseases attracts more attentions than before. Neutrophil elastase (NE), a powerful neutrol proteinase capable of causing major tissue destruction in many diseases, is mainly present in the azurophil granules of neutrophil. A solid-phase enzyme-linked immunosorbent-biotin assay for human NE has been developed. Only 20?l of plasma sample is needed. Non-smoking healthy adults have 33.7?6.7ng NE/ml plasma; patients with chronic obstructive pulmonary diseases, 48.0+ 10.9 ng NE/ml; patients with pyogenic dermatisis, 69.1?15.7 ng NE/ml; patients with systemic lupus erythematosus, 61.0?13.9 ng NE/ml; patients with aplastic anemia, only 12.3?5.3 ng NE/ml. All of these results have significant differences compared with the result of healthy adults. The results of assay showed that this quantitative assay with good specificity and accuracy may be a better criterion for investigating the effect of NE on pathogenesis of some diseases.

Purification and Charaterization of Human Neutrophil Defensins / 中国免疫学杂志

Defensins, a family of 3-4 KD cationic peptides, have been found recently in the cytoplasmicgranules of human, rabbit, guinea pig and rat polymorphonuclear neutrophils and rabbit alveolarmacrophages. The defence roles of these molecules, such as antimicrobial activity, viral inactiva-tion, tumor cell cytotoxicity, chemotactic and opsonic actions, etc. have been adequately studied.In order to investgate the role of Defensins in the neutrphil-mediated pathological process ofsome diseases, we isolated and purified human neutrophil Defensins. By using Sephadex G100and Bio-gel P10 gel filtration chromatography, the purified molecules were obtained. The puritywas identified by AU-PAGE,SDS-PAGE, and amino acid composition analysis. In vitro fugicidalactivity of the purified human neutrophil Defensins against Candida albicans was also examined.When incubated with 33 Mol concentration of human Defensins, more than 90% of Candida,al-bicans were killed in the methylene blue assay system.