ABSTRACT
ABSTRACT Purpose: To investigate human beta-defensins (HBDs) and cathelicidin LL-37 (LL-37) expressions in patients with pterygium. Methods: In this retrospective consecutive case series, 26 pterygium specimens and 15 normal conjunctival specimens of 15 control subjects were in vestigated. Expressions of HBD-1, HBD-2, HBD-3, and LL-37 were assessed using immuno histochemical staining. A brown color in the cytoplasm and/or nuclei of epithelial cells indicated positive staining for HBDs and LL-37. For each antibody, the intensity of the reaction (negative [-], weak [1+], moderate [2+], or strong [3+]) was determined to describe the immunoreactions. Results: The median age was 52 years in both groups. There were no significant differences in age and sex between the groups (p=0.583, p=0.355, respectively). Of the 26 pterygium specimens, 15 (57.7%) (14 weak, 1 moderate staining) showed HBD-2 expression, which was not observed in any of the control specimens. One (3.8%) pterygium and one (6.7%) control specimen demonstrated weak staining for HBD-3. HBD-2 expression was significantly higher in the pterygium specimens than in the controls (p=0.002). None of the tissue specimens had positive staining for HBD-1 or LL-37 in either group (both; p=1.00). Conclusions: HBD-2 expression was higher in pterygium specimens than in the controls. HBD-2 expression that might be stimulated by inflammatory cytokines may be related to inflammation and fibrovascular proliferation and may play a role in pterygium pathogenesis.
RESUMO Objetivo: Investigar as expressões beta defensinas humanas (HBD) e catelicidina em pacientes com pterígio. Métodos: Nesta série de casos retrospectivos consecutivos, 26 espécimes de pterígio e 15 espécimes conjuntivais normais de 15 indivíduos controle foram investigados. As expressões de HBD-1, HBD-2, HBD-3 e catelicidina (LL-37) foram avaliadas por coloração imuno-histoquímica. Uma cor castanha no citoplasma ou nos núcleos de células epiteliais foi definida como coloração positiva para HBDs e LL-37. Para cada anticorpo foi determinada a intensidade da reação (negativo [-], fraco [1+], moderado [2+] ou forte [3+]) para descrever as imunoreações. Resultados: A idade média foi de 52 anos em ambos os grupos. Não houve diferença significativa entre os grupos em termos de idade e sexo (p=0,583, p=0,355, respectivamente). Das 26 amostras de pterígio, 15 (57,7%) (14 fracas e 1 moderada) demonstraram a expressão de HBD-2 enquanto não foi encontrada em nenhum dos espécimes de controlo. Um dos pterígios (3,8%) e um dos espécimes de controlo (6,7%) demonstraram fraca coloração para HBD-3. A expressão de HBD-2 foi significati vamente maior nos espécimes de pterígio do que nos controles (p=0,002). Nenhum dos espécimes de tecido apresentou coloração positiva para HBD-1 ou LL-37 em ambos os grupos (ambos p=1,00). Conclusão: Encontramos aumento da expressão de HBD-2 em espécimes de pte rígio em relação aos controles. A expressão de HBD-2 que pode ser estimulada por citocinas inflamatórias pode estar relacionada com inflamação e proliferação fibrovascular e pode desempenhar um papel na patogênese do pterígio.
Subject(s)
Humans , Male , Female , Adult , Middle Aged , Aged , Pterygium/metabolism , Antimicrobial Cationic Peptides/analysis , beta-Defensins/analysis , Reference Values , Biopsy , Immunohistochemistry , Case-Control Studies , Retrospective Studies , Statistics, Nonparametric , Conjunctiva/chemistryABSTRACT
OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA). Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n = 61) than in mature milk samples (n = 39), as follows: median and range, 8.52 (2.6-16.3) µg/ml versus 0.97 (0.22-3.78), p < 0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs) of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL) was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients. .
OBJETIVO: Descrever a atividade antimicrobiana da defensina-beta 2 na glândula mamária e secretada no leite materno humano. MÉTODOS: A produção de peptídeos foi realizada por clonagem de DNA. Os níveis de defensina-beta 2 foram quantificados em 61 amostras de colostro e 39 de leite maduro de doadoras saudáveis pelo teste ELISA indireto. Por um ensaio de halo de inibição, avaliamos a atividade contra sete isolados clínicos diarreicos de crianças entre 0 e 2 anos. A atividade da defensina 2 contra três patógenos oportunistas que podem causar infecções nosocomiais foi determinada pelo teste de microdiluição. RESULTADOS: Os níveis de peptídeos estavam significativamente maiores nas amostras de colostro (n = 61) que de leite maduro (n = 39), como segue: 8,52 (2,6-16,3 µg/mL) mediana e faixa em comparação a 0,97 (0,22-3,78), p < 0,0001; teste de Mann-Whitney. O peptídeo recombinante foi obtido da alta atividade antimicrobiana demonstrada contra uma ampla gama de bactérias patogênicas. Sua atividade antibacteriana foi demonstrada em um disco contendo entre 1-4 µg, que produziu zonas de inibição entre 18 e 30 mm contra três isolados de Salmonella spp. e quatro de E. coli. A defensina-beta 2 demonstrou concentrações inibitórias mínimas (CIMs) de 0,25 µg/mL e 0,5 µg/mL para S. marcescen and P. aeruginosa, ao passo que uma CIM maior (4 µg/mL) foi obtida contra um isolado de cepa multirresistente de A. baumannii. CONCLUSÕES: Até onde sabemos, este estudo é o primeiro a relatar níveis de defensina em mulheres da América Latina. A produção e a atividade da defensina 2 no leite materno comprovam sua importância como uma molécula de defesa para a saúde intestinal em pacientes pediátricos. .
Subject(s)
Adult , Female , Humans , Pregnancy , Young Adult , Colostrum/chemistry , Milk, Human/chemistry , beta-Defensins/pharmacology , Enzyme-Linked Immunosorbent Assay/methods , Escherichia coli/drug effects , Lactation/immunology , Microbial Sensitivity Tests , Reverse Transcriptase Polymerase Chain Reaction/methods , Salmonella/drug effects , beta-Defensins/analysisABSTRACT
BACKGROUND: Recently, a continuous growth of interest has been observed in antimicrobial peptides (AMPs) in the light of an alarming increase in resistance of bacteria and fungi against antibiotics. AMPs are used as biomarkers in diagnosis and monitoring of oral cavity pathologies. Therefore, the determination of specific protein profiles in children diagnosed with early childhood caries (ECC) might be a basis for effective screening tests and specialized examinations which may enable progression of disease. METHODS: The objective of the studies was to determine the role of histatin-5 and ß-defensing-2 as a diagnostic marker of early childhood caries progression. In this work, results of concentration determination of two salivary proteins (histatin-5 and ß-defensin-2) were presented. In addition, bacterial profiles from dental plaque in various stages of ECC and control were marked. The assessment of alteration in the concentration of these two proteins in a study group of children with various stages of ECC and a control group consisting of children with no symptoms was performed by enzyme-linked immunosorbent assays. RESULTS: The statistical analysis showed a significant increase in the concentration of histatin-5 and ß-defensin-2 in the study group compared to the control group and correlated with the progression of the disease. CONCLUSIONS: The confirmation of concentration changes in these proteins during the progression of dental caries may discover valuable disease progression biomarkers.
Subject(s)
Humans , Male , Female , Child, Preschool , Child , Saliva/chemistry , beta-Defensins/analysis , Dental Caries/diagnosis , Histatins/analysis , Streptococcus/classification , Streptococcus/growth & development , Enzyme-Linked Immunosorbent Assay , Biomarkers/analysis , Colony Count, Microbial , Signal Transduction , Linear Models , Bacterial Typing Techniques , Disease Progression , Dental Caries/microbiology , Dental Caries Susceptibility , Early Diagnosis , Lacticaseibacillus rhamnosus/growth & development , Anti-Infective Agents/analysisABSTRACT
Mature mouse beta defensin 2 (mBD2) is a small cationic peptide with antimicrobial activity. Here we established a prokaryotic expression vector containing the cDNA of mature mBD2 fused with thioredoxin (TrxA), pET32a-mBD2. The vector was transformed into Escherichia Coli (E. coli) Rosseta-gami (2) for expression fusion protein. Under the optimization of fermentation parameters: induce with 0.6 mM isopropylthiogalactoside (IPTG) at 34ºC in 2×YT medium and harvest at 6 h postinduction, fusion protein TrxA-mBD2 was high expressed in the soluble fraction (>95 percent). After cleaved fusion protein by enterokinase, soluble mature mBD2 was achieved 6 mg/L with a volumetric productivity. Purified recombinant mBD2 demonstrated clear broad-spectrum antimicrobial activity for fungi, bacteria and virus. The MIC of antibacterial activity of against Staphylococcus aureus was 50 µg/ml. The MIC of against Candida albicans (C. albicans) and Cryptococcus neoformans (C. neoformans) was 12.5µg/ml and 25µg/ml, respectively. Also, the antimicrobial activity of mBD2 was effected by NaCl concentration. Additionally, mBD2 showed antiviral activity against influenza A virus (IAV), the protective rate for Madin-Darby canine kidney cells (MDCK) was 93.86 percent at the mBD2 concentration of 100 µg/ml. These works might provide a foundation for the following research on the mBD2 as therapeutic agent for medical microbes.