Inhibition of defensin A and cecropin A responses to dengue virus 1 infection in Aedes aegypti / Inhibición de las respuestas de defensina A y cecropina A contra la infección del virus dengue 1 en Aedes aegypti

Abstract | Introduction: It is essential to determine the interactions between viruses and mosquitoes to diminish dengue viral transmission. These interactions constitute a very complex system of highly regulated pathways known as the innate immune system of the mosquito, which produces antimicrobial peptides that act as effector molecules against bacterial and fungal infections. There is less information about such effects on virus infections. Objective: To determine the expression of two antimicrobial peptide genes, defensin A and cecropin A, in Aedes aegypti mosquitoes infected with DENV-1. Materials and methods: We used the F1 generation of mosquitoes orally infected with DENV-1 and real-time PCR analysis to determine whether the defensin A and cecropin A genes played a role in controlling DENV-1 replication in Ae. aegypti. As a reference, we conducted similar experiments with the bacteria Escherichia coli. Results: Basal levels of defensin A and cecropin A mRNA were expressed in uninfected mosquitoes at different times post-blood feeding. The infected mosquitoes experienced reduced expression of these mRNA by at least eightfold when compared to uninfected control mosquitoes at all times post-infection. In contrast with the behavior of DENV-1, results showed that bacterial infection produced up-regulation of defensin and cecropin genes; however, the induction of transcripts occurred at later times (15 days). Conclusion: DENV-1 virus inhibited the expression of defensin A and cecropin A genes in a wild Ae. aegypti population from Venezuela.

Resumen | Introducción. Es esencial determinar las interacciones entre los virus y los mosquitos para disminuir la transmisión viral. Estas interacciones constituyen un sistema muy complejo y muy regulado conocido como sistema inmunitario innato del mosquito, el cual produce péptidos antimicrobianos, moléculas efectoras que funcionan contra las infecciones bacterianas y fúngicas; se tiene poca información de su acción sobre los virus. Objetivo. Determinar la expresión de dos genes AMP (defensina A y cecropina A) en mosquitos Aedes aegypti infectados con el virus DENV-1. Materiales y métodos. Se infectaron oralmente mosquitos de generación F1 con DENV-1 y mediante el análisis con PCR en tiempo real se determinó el potencial papel de los genes defensina A y cecropina A en el control de la replicación del DENV-1 en Ae. aegypti. Como referencia, se infectaron mosquitos con Escherichia coli. Resultados: Los mosquitos no infectados expresaron niveles basales de los ARNm de los genes defensina A y cecropina A en diversos momentos después de la alimentación. Los mosquitos infectados experimentaron una reducción, por lo menos, de ocho veces en la expresión de estos ARNm con respecto a los mosquitos de control en todo el periodo posterior a la alimentación. En contraste con el comportamiento del virus DENV-1, los resultados mostraron que la infección bacteriana produjo una regulación positiva de los genes defensina y cecropina; sin embargo, la inducción de los transcritos ocurrió tardíamente (15 días). Conclusión. El virus DENV-1 inhibió la expresión de los genes defensina A y cecropina A en una población silvestre de Ae. aegypti en Venezuela.

Serum defensin levels in patients with systemic sclerosis

Abstract Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and lung as well as involvement of kidney, gastrointestinal system and heart. Aetiology and exact mechanism of disease is poorly understood. The association between antimicrobial peptides (AMPs) and other diseases such as idiopathic pulmonary fibrosis, diffuse panbronchiolitis, pulmoner alveolar proteinosis and psoriasis have been reported. A small number of studies have examined the role of AMPs on autoimmune diseases which has not been studied in scleroderma yet. We aimed to investigate AMP serum levels and their association with disease characteristics of SSc. Methods: Forty-two patients (40 female, mean age 42 years) and 38 healthy subjects (32 female, mean age 38 years) were enrolled. For SSc patients, the following data were recorded: disease subset (limited/diffuse), autoantibodies (antinuclear, anti-centromere (ACA), and anti-SCL-70), blood tests, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), modified Rodnan skin score, presence and history of digital ulcers, kidney, gastrointestinal disease and lung involvement assessed by computed tomography and pulmonary function tests. Association between serum AMPs and disease characteristics were analysed. Results: Twenty-nine of the patients had diffuse (69%) and 13 of the patients had limited (31%) systemic sclerosis. Average disease duration was 5.5 years. Pulmonary involvement was detected in 20 patients (47.6%). Serum concentration of alpha defensin was higher than healthy subjects (563 ± 415 vs 377 ± 269 ng/mL, p = 0.02). However, no difference was observed for beta-1 and beta-2 defensins in SSc patients and healthy controls. In sub-group analysis patients with interstitial lung disease had higher levels of alpha defensin than those without lung involvement (684 ± 473 vs 430 ± 299 ng/ml, p = 0.04). There was also correlation between alfa defensin serum concentrations and CRP (r = 0.34). Conclusions: Alpha defensin levels are increased in scleroderma patients and correlated with lung involvement indicating a role in the pathogenesis of disease. Trial registration: This study is not a clinical trial study.(AU)

¿Qué hay de nuevo para el diagnóstico de infecciones periprotésicas después del Consenso de Filadelfia? / What's new for the diagnosis of periprosthetic infections after the Philadelphia consensus?

Resumen: Actualmente contamos con diversos métodos de laboratorio para el diagnóstico de las infecciones periprotésicas, algunos ampliamente probados y otros en estudio. Han aparecido nuevos biomarcadores después del Consenso de Filadelfia, por tal motivo, nos planteamos hacer una revisión acerca de qué hay de nuevo para su diagnóstico después del Consenso y cuáles podrían ser los más útiles para el trabajo clínico diario. Material y métodos: Se revisaron artículos publicados entre 2013-2017 en cinco revistas de alto impacto. Las variables fueron: tipo de biomarcador, cifras de corte, sensibilidad, especificidad, valor predictivo positivo, valor predictivo negativo, área bajo la curva, razón de momios diagnósticos y cocientes de probabilidad positivos y negativos. Se calificó nivel de evidencia. Resultados: Los resultados se agruparon en Tablas. Se encontraron 54 artículos, de los cuales 31 no se ajustaban a los criterios de inclusión y fueron excluidos; sólo se incluyeron 23. Se encontraron 19 biomarcadores, cinco de los cuales no habían sido reportados hasta antes de 2013: La α defensina sinovial 1, la β defensina humana 3, el lactato sinovial y los receptores tipo Toll 1 y Toll 6. Conclusión: Los biomarcadores que ofrecen mayor utilidad clínica para el diagnóstico de IAP son: la proteína C reactiva, la esterasa leucocitaria, la interleucina-6, la interleucina-1β, la α-defensina y la interleucina-17. Detectamos cinco nuevos marcadores. Los estudios analizados muestran heterogeneidad en sensibilidad, especificidad y en sus cifras de corte. En la mayoría no usan aplicaciones estadísticas avanzadas que los harían más confiables.

Abstract: We now have a great variety of laboratory diagnostic tools, for the detection of PJI, some of them widely used and others under study. After the Philadelphia Consensus, they have emerged some new biomarkers. Because of that, we consider useful to review which new biomarkers we have for the diagnosis of PJI after the Consensus and which of them could be more useful in daily clinic work. Material and methods: We searched for articles published from 2013 to 2017 in 5 high impact journals. The analized variables were: biomarker type, cutoff value, sensitivity, specificity, positive predictive value, negative predictive value, area under the curve, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio. We value their evidence level. Results: Results were grouped in Tables. They were found 54 articles, 31 of them didn't meet the inclusion criteria so they were excluded; 23 studies were included in the revision. We found a total of 19 biomarkers studies, 5 of them weren't reported before 2013: Sinovial α defensin 1, human β defensin-3, sinovial lactate and Toll-like receptors 1 and 6. Conclusion: Of all the markers reviewed for the diagnosis of PJI, C reactive protein, esterase test strip, interleukin-6, interleukin-1 β, α defensin and interleukin-17 show the highest diagnostic utility. We found 5 new markers. The articles studies show high heterogeneity in their reported sensitivity, specificity and cutoff values. In most of them were not used advanced statistical tools which could make them more reliable.

Tuberculin skin test and interferon-gamma release assay values are associated with antimicrobial peptides expression in polymorphonuclear cells during latent tuberculous infection

It has been reported that patients with progressive tuberculosis (TB) express abundant amounts of the antimicrobial peptides (AMPs) cathelicidin (LL-37) and human neutrophil peptide-1 (HNP-1) in circulating cells, whereas latent TB infected donors showed no differences when compared with purified protein derivative (PPD) and QuantiFERON®-TB Gold (QFT)-healthy individuals. The aim of this study was to determine whether LL-37 and HNP-1 production correlates with higher tuberculin skin test (TST) and QFT values in TB household contacts. Twenty-six TB household contact individuals between 26-58 years old TST and QFT positive with at last two years of latent TB infection were recruited. AMPs production by polymorphonuclear cells was determined by flow cytometry and correlation between TST and QFT values was analysed. Our results showed that there is a positive correlation between levels of HNP-1 and LL-37 production with reactivity to TST and/or QFT levels. This preliminary study suggests the potential use of the expression levels of these peptides as biomarkers for progression in latent infected individuals.

Impact and potential mechanism of human α-defensin 1 on low-density lipoprotein oxidation ability of ECV304 cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To explore the role and potential mechanism of human α-defensin 1 (HNP-1) on low-density lipoprotein (LDL) oxidation ability of human endothelial cells (EVC304).</p><p><b>METHODS</b>Post incubation with LDL for 3 h, the malondialdehyde (MDA) and protein carbonyl (PCO) were detected in untreated ECV304 (control) and in HNP-1 transfected ECV304 in the presence and absence of siRNA against HNP-1. Flow cytometry and fluorescence microscopy were used to detect the generation of oxygen free radical in the ECV304 which have been pretreated by LDL, LPS and HNP-1, respectively.</p><p><b>RESULT</b>Compared with control group, MDA level was significantly increased in HNP-1 transfected [(4.21 ± 0.03) vs. (3.15 ± 0.02) nmol/mg · pro] or in HNP-1 stimulated ECV304 cells [(14.49 ± 1.10) vs. (9.47 ± 1.18) nmol/mg · pro], which could be significantly downregulated by siRNA [(3.76 ± 0.48) vs. (4.54 ± 0.28) nmol/mg·pro, all P < 0.05]. PCO was also significantly increased in HNP-1 transfected ECV304 cells. The levels of free radical were significantly increased in HNP-1 transfected or HNP-1 stimulated ECV304 cells.</p><p><b>CONCLUSION</b>HNP-1 can enhance the LDL oxidation ability of human endothelial cells via promoting the generation of free radicals.</p>

Diagnostic value of human neutrophil peptide in spontaneous bacterial peritonitis / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the correlation between human neutrophil peptide (HNP) and spontaneous bacterial peritonitis (SBP) in order to assess the diagnostic value of quantitative measurement of these alpha-defensins.</p><p><b>METHODS</b>Seventy-seven patients with cirrhosis and ascites were divided into two groups according to diagnosis of SBP (n = 45 with SBP and n = 32 without SBP). Twenty-eight healthy individuals were analyzed as controls. HNP was detected by double-antibody sandwich assay. Peripheral white blood cell (WBC) counts, neutrophil ratio, and levels of procalcitonin (PCT) and C-reactive protein (CRP) were determined by standard methods. Receiver operating characteristic (ROC) curves were used to compare the diagnostic values of HNP, PCT and CRP in SBP.</p><p><b>RESULTS</b>There were no significant differences between the three groups (SBP, non-SBP, and healthy controls) for WBC count ((6.01+/-2.25)*109 /L, (4.85+/-1.94)*109 /L, and (5.49+/-1.76)*109 /L; F = 2.91, P more than 0.05) and neutrophil ratio (70.70%+/-10.42%, (68.20%+/-8.97%, and 69.50%+/-8.69%; F = 3.07, P more than 0.05). However, compared to the non-SBP group and the healthy controls, the SBP group showed significantly higher levels of HNP ((9.99+/-3.33) ng/ml and (8.92+/-2.30) ng/ml vs. (17.66+/-6.40) ng/ml; q = 3.20 vs. 3.52, P less than 0.05), serum CRP ((15.08+/-9.95) ng/ml and (5.96+/-2.91) ng/ml vs. (31.32+/-18.65) mg/L; q = 11.03 vs. 3.69, P less than 0.05), and positive rate of PCT (25.0% and 10.0% vs. 62.2%; X2 = 10.41 vs. 15.40, P less than 0.0125). The areas under the curve (AUC) showed the following descending trend: HNP more than PCT more than CRP (0.719, 0.707, and 0.629 respectively). Using cut-off points of 10 ng/ml for HNP, 0.5 ng/ml for PCT, and 8 mg/L for CRP, the respective sensitivities for diagnosis of SBP were 71.1%, 62.2%, and 73.3%, the respective specificities were 71.9%, 75.0%, and 56.3%, and the respective Youden's indexes were 0.430, 0.372, and 0.296.</p><p><b>CONCLUSION</b>HNP is closely related to SBP and can diagnose SBP as reliably as PCT. CRP may help to diagnose SBP, but the results from routine blood testing did not show sufficient statistical significance for diagnosing SBP.</p>

Antimicrobial peptides and nitric oxide production by neutrophils from periodontitis subjects

Neutrophils play an important role in periodontitis by producing nitric oxide (NO) and antimicrobial peptides, molecules with microbicidal activity via oxygen-dependent and -independent mechanisms, respectively. It is unknown whether variation in the production of antimicrobial peptides such as LL-37, human neutrophil peptides (HNP) 1-3, and NO by neutrophils influences the pathogenesis of periodontal diseases. We compared the production of these peptides and NO by lipopolysaccharide (LPS)-stimulated neutrophils isolated from healthy subjects and from patients with periodontitis. Peripheral blood neutrophils were cultured with or without Aggregatibacter actinomycetemcomitans-LPS (Aa-LPS), Porphyromonas gingivalis-LPS (Pg-LPS) and Escherichia coli-LPS (Ec-LPS). qRT-PCR was used to determine quantities of HNP 1-3 and LL-37 mRNA in neutrophils. Amounts of HNP 1-3 and LL-37 proteins in the cell culture supernatants were also determined by ELISA. In addition, NO levels in neutrophil culture supernatants were quantitated by the Griess reaction. Neutrophils from periodontitis patients cultured with Aa-LPS, Pg-LPS and Ec-LPS expressed higher HNP 1-3 mRNA than neutrophils from healthy subjects. LL-37 mRNA expression was higher in neutrophils from patients stimulated with Aa-LPS. Neutrophils from periodontitis patients produced significantly higher LL-37 protein levels than neutrophils from healthy subjects when stimulated with Pg-LPS and Ec-LPS, but no difference was observed in HNP 1-3 production. Neutrophils from periodontitis patients cultured or not with Pg-LPS and Ec-LPS produced significantly lower NO levels than neutrophils from healthy subjects. The significant differences in the production of LL-37 and NO between neutrophils from healthy and periodontitis subjects indicate that production of these molecules might influence individual susceptibility to important periodontal pathogens.

Correlation of DEFA1 Gene Copy Number Variation with Intestinal Involvement in Behcet's Disease

Copy number variation has been associated with various autoimmune diseases. We investigated the copy number (CN) of the DEFA1 gene encoding alpha-defensin-1 in samples from Korean individuals with Behcet's disease (BD) compared to healthy controls (HC). We recruited 55 BD patients and 35 HC. A duplex Taqman(R) real-time PCR assay was used to assess CN. Most samples (31.1%) had a CN of 5 with a mean CN of 5.4 +/- 0.2. There was no significant difference in the CN of the DEFA1 gene between BD patients and HC. A high DEFA1 gene CN was significantly associated with intestinal involvement in BD patients. Variable DEFA1 gene CNs were observed in both BD patients and HC and a high DEFA1 gene CN may be associated with susceptibility to intestinal involvement in BD.

Comparison of neutrophil apoptosis alpha-defensins and calprotectin in children with and without severe early childhood caries

The infectious nature of severe early-childhood caries[S-ECC] points to the possible participation of immunologic host responses including neutrophils and their antimicrobial products. The aim of this study was to determine the neutrophil apoptosis, alpha-defensins [HNP1-3] and calprotectin levels in the saliva of preschool children and the association with S-ECC. Oral examinations were performed on 87 children aged 3-5 years and non stimulated whole saliva samples were collected. Thirty of these subjects were considered S-ECC children, 30 with moderate caries [MC] and 27 were caries free [CF]. to detect apoptosis, cell staining was done with Annexin-V-Fluos and propidium iodide, and they were analyzed by fluorescent microscopy. The concentration of alpha-defensins and calprotectin were assessed using ELISA. There were no statistical differences between groups considering the HNP1-3 or calprotectin salivary levels[p=0.06 and p=0.23, respectively]. The HNP1-3 and calprotectin levels were negatively correlated and the correlation was significant in MC group [p=0.03]. Lower levels of apoptotic neutrophils were obtained from CF subjects as compared with S-ECC children [p=0.03]. Our findings establish that apoptotic mechanisms could be implicated in the immunity responses associated with S-ECC. We cannot yet determine if the level of salivary alpha-defensins or calprotectin is predictive of S-ECC

Péptidos antimicrobianos: [revisión] / Antimicrobial peptides: [review]

Los péptidos antimicrobianos son las moléculas efectoras del sistema inmune innato, cuyas familias se encuentran en casi todos los organismos, desde bacterias hasta mamíferos. Son una familia de sustancias polifacéticascon complejos mecanismos deacción relacionados con la interacción con el patógeno a través de su membrana, o afectando blancos internos, como la replicación del ADN y la síntesis de proteínas, e interactuando con el huésped con funciones inmunomoduladoras de la regulación delproceso inflamatorio y de la cicatrización. Aunque la generación de resistencia a los péptidos antimicrobianos es mucho menorsi se compara con la generada por losantibióticos convencionales, existen mecanismos de resistencia ya descritos, como la degradación por proteasas, la liberación de proteínas inhibidoras o los cambios en la conformación de la membrana externa del patógeno. El estudio de estas sustancias hapermitido evidenciar sus usos potenciales en el ámbito clínico para contrarrestar los inconvenientes de la resistencia a los antibióticos; sin embargo, a pesar de los grandesavances logrados en este campo, aún quedan puntos controversiales por dilucidar.

The antimicrobial peptides (AMP) are theeffectors molecules of the innate immunesystem, finding groups of this kind of substances in almost all living organisms from bacteria to mammals. They are a family of versatile substances with complexes action mechanisms in the pathogen they interact with membrane, DNA synthesis and protein synthesis and folding, and also with the hostshowing immunomodulatory functions inwound healing and inflammation process.Even though the generation of resistance to the AMP is lower compare with conventional antibiotics there are resistance mechanism already describe to this kind of substances like degradation by proteases, releasing ofinhibitory substances or conformationalchanges in the external membrane of thepathogen. Actually the study of this group of substances has make them see as potential tools for clinical use helping to coun-teract the problem of antibiotic resistance, but even great progress had been made in this field there still exist some controversial issues for future study.

Role of Toll-like receptor 4 and human defensin 5 in primary endocervical epithelial cells / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endocervical epithelial cells play early roles in the defense of upper female genital tract to pathogens. Toll-like receptors (TLRs) and human defensins (HD) have recently been identified as fundamental components of the innate immune responses to bacterial pathogens. We aimed to use in vitro model of human primary endocervical epithelial cells (HPECs) to investigate their roles in innate immune response of the endocervix.</p><p><b>METHODS</b>TLR4 expression and distribution in HPECs and endocervix were investigated by immunofluorescence (IF). Cultured HPECs were divided into lipopolysaccharide (LPS) group which were treated by LPS for 0, 24 and 48 hours, and control group without treatment. At each time point, the levels of HD5, IL-6 and TNF-alpha in supernants were determined by ELISA. TLR4 and HD5 expressions of cells were detected by Western blotting simultaneously. HD5 expression pattern was also compared between the HeLa cell line and HPECs.</p><p><b>RESULTS</b>Endocervix tissue surface and HPECs expressed TLR4. After incubated with LPS, HPECs expressed significantly higher levels of TLR4 than control group, especially after 24 hours (P < 0.01), however decreased after 48 hours with a similar level of TLR4 expression compared with control group. LPS could upregulate the secretion of HD5, IL-6 and TNF-alpha in a time-dependent manner (24 hours: P < 0.05; 48 hours: P < 0.01, compared with control group). Intracellular HD5 expression levels decreased over time. HD5 expression patterns in HPECs were different from HeLa cell line.</p><p><b>CONCLUSIONS</b>To respond to LPS stimulation, HPECs may function in the mucosal immune defense through TLR4 activation and HD5 secretion. HPEC is considered a significant model for immunological study.</p>

Effect of inhibitor HNP1 transfection on tumor growth of human nasopharyngeal carcinoma cell line HNE-1 / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#The aim of this study is to investigate the effects of inhibitor HNP1 transfection on proliferation and tumor growth of human nasopharyngeal carcinoma cell line HNE1.@*METHOD@#HNE1 cells were transfected with HNP1 by liposome, and the cytotoxic effect was detected by MTT, In vivo tumor growth test was performed in nude mice inoculated with transfected HNE1 cells. The therapeutic effect of HNP1 was evaluated in the inoculated tumors, alpha-defensin 1 expression was detected in implanted tumor tissues by immunohistochemical stain.@*RESULT@#HNP1 transfection significantly inhibited the proliferation of HNE1 cells. MTT assay confirmed cytotoxic effect. In vivo study showed tumor volume was significantly smaller in HNP1 transfection group than that in control group (P < 0.01). Immunohistochemical stain showed alpha-defensin expression was increased in HNP1 transfection group.@*CONCLUSION@#HNP1 transfection can inhibit the proliferation of HNE1 cells, as well as tumor growth.

Soluble expression and purification of human alpha-defensin-5 in Escherichia coli / 生物工程学报

DNA fragment containing human alpha-defensin 5 mature peptide (mHD-5) coding sequence with biased codons of E. coli was amplified by PCR, which was subsequently cloned into the plasmid pMAL-p2x in order to create pMAL-p2x-mHD-5 expression vector. The plasmid pMAL-p2x-mHD-5 was transferred into engineered strain BL21(DE3) to express heterogeneous fusion protein (MBP-mHD-5). The soluble MBP-mHD-5 targeted protein inducible expressed by IPTG was accounted for about 30% under optimized conditions. The recombinant mHD-5 (rmHD-5) peptide was successfully purified through a separation process including affinity chromatography, Factor Xa digestion and ion exchange chromatography. The bioactivity of rmHD-5 was examined by bacteria-inhibition tests in liquid culture. The growth of E. coli ATCC25922 was dramatically suppressed with an inhibition rate of 90%, with the presence of 62.5 microg/mL rmHD-5 in the media. These results indicate that the strategy of soluble expression of fusion protein in E. coli can be a useful and practical way to produce bioactive defensins.

Inflammation and neovascularization in diabetic atherosclerosis.

Diabetes mellitus, the major cardiovascular risk factor, accentuates the inflammation and neovascularization processes leading to enhanced progression of atherosclerotic complications. Inflammation in diabetes mellitus is the key initiator of atherosclerotic process, which results in acute coronary events. Atherosclerosis evolves from the endothelial cell dysfunction and succeeding entry of hemodynamically derived leukocytes by migration, activation and production of lipid gruel leading to atheromatous plaque progression and subsequent regression. Diabetic plaque progression is associated with increased neovascularization, which is a nature's compliment in the sustenance of plaque growth by its nutrient supply. Neovessels may act as conduit for lipid debridment and alternative channel for inflammatory process. In addition, neovascularization induces intra-plaque hemorrhage due to the fragility of the neovessels and associated inflammation, resulting in plaque instability. The intra-plaque hemorrhage is a detrimental base, which begets the progress of atheroma by inducing oxidative stress and endothelial dysfunction. Intra-plaque hemorrhage is increased in diabetes with an associated increase in hemoglobin-haptoglobin complex (Hb-Hp2-2), which further induces oxidative stress and endothelial cell dysfunction. We conclude that inflammation and neovascularization of the plaque may act as major mechanism augmenting plaque instability in diabetes mellitus.

Characterization of polypeptides of vernix caseosa [VC] and Its role on the neonate skin surfsce as an innate antimicrobial barrier

Vernix caseosa [VC] is a greasy substance, that gradually covers the fetal skin during the last trimester of pregnancy. The vernix had been shown to have antimicrobial properties. Given the strategic position of vernix [a relatively unstudied material] on the fetal skin surface, this study aimed to investigate whether natural antimicrobial peptides/proteins contribute to surface protection of newborns at birth, hence the significant basis for leaving vernix in place at birth was provided. This study characterized peptides and proteins in vernix with emphasis on antimicrobial activity. Concentrated peptide/protein extracts were obtained from samples of vernix from full-term healthy newborn infants and screened for antimicrobial activity using inhibition zone assays. Proteins/peptides fraction in all vernix extracts exhibited a clear and strong antibacterial activity [inhibition of growth on nutrient agar] against Klebsiella pneumoniae, Staphylococcus aureus and Streptococcus pyogenes, in addition to antifungal activity against Candida albicans. Alpha- defensins [HNP1-3], LL-37 and lysozyme were characterized among the active components. In addition, psoriasin and ubiquitin were identified in fractions derived from vernix, that exhibited antibacterial activity

Expression of Human Beta-Defensin in Human Male Urogenital Organs / 감염

BACKGROUND: Defensins are small (3.5~5 kDa) cationic antimicrobial peptides that have a broad spectrum of activity that includes gram-negative bacterias, yeasts and enveloped viruses. The defensins contain six cysteine residues forming three disulfide bridges depending on the spacing of the cysteine residues and the connectivity of the disulfide bridge, defensins are classified into two families, the alpha-defensins (HNP) and beta-defensins (HBD). Recently two human epithelial beta defensins, HBD-1 and HBD-2 have been identified. HBD-1 has been detected in a number of normal mucosal sites, but HBD-2 is highly restricted in its expression by inflammatory stimulations. we invesigated the expression of hunam beta defensin in human male urogenital organs. METHODS: Specimens of normal human male testis, epididymis, prostate, seminal vesicles, vas deferens, urethra, bladder, ureter, kidney, pyelonephritis, epididymitis, clear renal cell carcinoma and transitional cell carcinoma of bladder were obtained as discarded material from urological surgery. Each sample was stored at snap frozen in liquid nitrogen subsequent to RNA extraction. Reverse transcription polymerase chain reaction (RT-PCR) was used to semiquantitate HBD-1 and HBD-2 mRNA using the housekeeping gene beta-actin as an internal control. Southern blotting and sequencing showed HBD-1, 2 expressions in male urogenital organs. RESULTS: We checked the expression of HBD-1, 2 mRNA in all specimen of normal human male urogenital organ, pyelonephritis, epididymitis, clear renal cell carcinoma and transitional cell carcinoma of bladder by RT-PCR and southern blotting analysis. We checked the homolgy of HBD-1, 2 by bands sequencing. CONCLUSION: Our study indicated that the normal male urogenital organs, infection and neoplasm in male urogenital organs expresses antimicrobial peptides. These may play an important role in the prevention of infections by bacterias, antimicrobial effects in infection and anticancer effects in neoplasm of male urogenital organs. These natural endogenous antibiotic peptides could be developed as novel therapeutic agents for fighting infections and neoplasms of the human male urogenital organs.