Clinical study of serum human-βeta-defensin-1 level for evaluating short-term prognosis in patients with acute-on-chronic liver failure / 中华肝脏病杂志

Objective: To evaluate the diagnostic value of serum human-βeta-defensin-1 level (HBD-1) for short-term (28-day) prognosis in patients with acute-on-chronic liver failure (ACLF). Methods: Fifty cases diagnosed with ACLF were selected. 20 cases with decompensated cirrhosis and 20 cases with compensated cirrhosis who were admitted at the same time were included. Age, gender, serum HBD-1 level, C-reactive protein (CRP), procalcitonin (PCT), neutrophil count/lymphocyte ratio (NLR), blood routine, coagulation function, liver function, kidney function, and other indicators from the three groups of patients were collected. Patients with ACLF were screened for indicators related to the short-term (28-day) prognosis. Patients were divided into an improvement group and a worsening group according to the 28-day disease outcome. The serum HBD-1 level and other above-mentioned indicators were compared between the two patient groups. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of serum HBD-1 levels for short-term prognosis in patients with ACLF. PCT, NLR, and prothrombin activity (PTA) application as a mono indicator and HBD-1 in combination with NLR, PCT, and PTA were compared to evaluate diagnostic efficacy for short-term prognosis in patients with ACLF. The intergroup mean of measurement data was determined using a t-test or analysis of variance. χ (2) test was used for comparison of count data. Spearman's rank correlation analysis was used for correlation analysis. Results: There was no statistically significant difference in age and gender among the three groups: ACLF, decompensated cirrhosis, and compensated cirrhosis (P > 0.05). The expression levels of serum HBD-1 in the ACLF group, decompensated cirrhosis group, and compensated cirrhosis group were (319.1 ± 44.4) ng/ml, (264.5 ± 46.5) ng/ml and (240.1 ± 35.4) ng/ml, respectively, while the ACLF group expression levels were significantly increased, with statistical significance (P < 0.01).The serum HBD-1 level was significantly higher in the ACLF worsening group (346.2 ± 43.6) ng/ml than that in the improvement group (308.5 ± 40.6) ng/ml, and the difference was statistically significant (P < 0.05). Correlation analysis showed that HBD-1, NLR, PCT, prothrombin time (PT), and international standardized ratio (INR) were negatively correlated with the 28-day disease outcome (improvement) of patients (P < 0.05). PTA was positively correlated with 28-day disease outcome (improvement) (P < 0.05). The area under the receiver operating characteristic curve (AUC) for evaluating HBD-1's diagnostic efficacy for short-term prognosis in patients with ACLF was 0.774, with a sensitivity of 0.750, a specificity of 0.786, and a cut-off point of 337.96 ng/ml. PCT, NLR, and PTA had greater diagnostic efficacy. HBD-1 combined with PTA had the highest diagnostic efficacy, with an AUC of 0.802, a sensitivity of 0.778, and a specificity of 0.786. The diagnostic efficacy of HBD-1+PCT, HBD-1+NLR and HBD-1, PCT, and NCR was superior to PTA mono. Conclusion: The serum HBD-1 level gradually increases with the aggravation of liver function injury and is negatively correlated with the short-term prognosis in patients with ACLF. Serum HBD-1 level has high sensitivity and specificity in predicting short-term prognosis in patients with ACLF, and its diagnostic efficacy is superior to that of PCT, NLR, and PTA. The combined application of HBD-1 and PTA has higher diagnostic efficacy; however, when the serum HBD-1 level is greater than 337.96ng/ml, it indicates poor prognosis in patients.

Defensin c-thionin from Capsicum chinense improves butyrate cytotoxicity on human colon adenocarcinoma cell line Caco-2

BACKGROUND: Butyrate is a histone deacetylase inhibitor that induces apoptosis and inhibits cell proliferation of colorectal cancer cells. To improve its anticancer activity, butyrate has been evaluated mixed with drugs and different molecules. Plant antimicrobial peptides are attractive anticancer alternative molecules because they show selective cytotoxic activity against different cancer cell lines. In this work, we explore if the plant defensin c-thionin (Capsicum chinense) can improve butyrate activity on Caco-2 cell line and we also determined the mechanism of death activated. RESULTS: The combined treatment of c-thionin (3.5 mM) and butyrate (50 mM) showed higher cytotoxicity on Caco-2 cells with respect to single treatments. Also, the combined treatment reduced cell proliferation and exhibited a higher rate of apoptosis than single treatments. Combined treatment induced caspases 8 and 9 activation to an extent comparable with that of butyrate while c-thionin did not activate caspases. Additionally, reactive oxygen species generation preceded the onset of apoptosis, and superoxide anion production was higher in cells treated with the combined treatment. CONCLUSIONS: The c-thionin from Habanero chili pepper improved the butyrate cytotoxicity on Caco-2 cells. This effect occurred through apoptosis induction associated with reactive oxygen species production. Therefore, the combination of butyrate with cytotoxic antimicrobial peptides could be an attractive strategy for cancer therapy.

Recombinant expression of Crassostrea gigas defensin in Pichia pastoris and its antibacterial activity / 生物工程学报

Defensins are endogenous cationic antimicrobial peptides rich in arginine and cysteine residues. They are important immune factors resisting pathogenic bacteria infection for mollusks. The 43 amino acid residues near the carboxyl terminal for Crassostrea gigas defensin (CgD) form its mature peptide region, responsible for the biological activity of CgD. First, two target genes, CgDH⁺ (with 6×His-tag at 3' end) and CgDH- (without 6×His-tag at 3' end) were separated and amplified by RT-PCR with specific primers from Crassostrea gigas mantle. These two target genes were ligated to the expression vector pPICZαA to construct recombinant expression vectors, pPICZαA-CgDH⁺ and pPICZαA-CgDH-, which were transformed into competent Pichia pastoris X-33 cells by electroporation respectively. The recombinant target proteins, CgDH⁺ and CgDH-, were induced for 72 h with 1% methanol at 29 °C and 250 r/min. The recombinant CgDH⁺ (5.78 kDa) was purified by immobilized metal affinity chromatography (IMAC), and identified by MALDI-TOF-TOF analysis, demonstrating that it was the expected target protein. Based on the concentration of the purified product, the estimated yield of recombinant CgDH⁺ was 2.32 mg/L. Antimicrobial assay showed that the culture medium supernatant containing recombinant CgDH⁺ and recombinant CgDH-, respectively, had activities against Staphylococcus aureus and Pseudomonas aeruginosa, indicating that the existence of 6×His tag in the recombinant proteins do not affect their biological activities.

Towards the Application of Human Defensins as Antivirals

Defensins are antimicrobial peptides that participate in the innate immunity of hosts. Humans constitutively and/or inducibly express α- and β-defensins, which are known for their antiviral and antibacterial activities. This review describes the application of human defensins. We discuss the extant experimental results, limited though they are, to consider the potential applicability of human defensins as antiviral agents. Given their antiviral effects, we propose that basic research be conducted on human defensins that focuses on RNA viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), respiratory syncytial virus (RSV), and dengue virus (DENV), which are considered serious human pathogens but have posed huge challenges for vaccine development for different reasons. Concerning the prophylactic and therapeutic applications of defensins, we then discuss the applicability of human defensins as antivirals that has been demonstrated in reports using animal models. Finally, we discuss the potential adjuvant-like activity of human defensins and propose an exploration of the ‘defensin vaccine’ concept to prime the body with a controlled supply of human defensins. In sum, we suggest a conceptual framework to achieve the practical application of human defensins to combat viral infections.

Péptidos antimicrobianos y mediadores lipídicos: rol en las enfermedades periodontales / Antimicrobial peptides and lipid mediators: their role in periodontal diseases

En la actualidad existe consenso en que el daño de los tejidos de soporte dentario que se produce durante la periodontitis es un proceso complejo en el cual la presencia de los patógenos periodontales es necesaria, pero no suficiente, para explicar en su totalidad la extensión y severidad de dicho daño. Asimismo, la destrucción del tejido de soporte periodontal es en gran medida producida por el desbalance de la respuesta inmune generada por el paciente frente a antígenos y factores de virulencia derivados de los patógenos periodontales. Esta respuesta inmune, desencadenada por las bacterias periodontopatógenas, incluye tanto mecanismos asociados a inmunidad innata como adaptativa, siendo el rol de los péptidos antimicrobianos y mediadores lipídicos aspectos relacionados con ambas ramas de la inmunidad y que no han sido completamente dilucidados en relación con sus mecanismos de acción contra los patógenos periodontales. En esta revisión se describe el rol de los péptidos antimicrobianos y de los mediadores lipídicos en la enfermedad periodontal, enfocándonos en su contribución tanto a la protección como a la destrucción del tejido de soporte dentario durante la infección periodontal. Se destaca además la importancia de considerarlos dentro del complejo escenario de la respuesta inmune durante las enfermedades periodontales, ya que forman parte fundamental de la respuesta inmune del hospedero. Analizar la enfermedad periodontal ampliando la perspectiva de estudio a este tipo de moléculas que participan de la respuesta inmune permitiría en el futuro lograr un nuevo enfoque terapéutico de las enfermedades periodontales.

Currently, there is consensus that the damage of the tooth support tissues that occurs during periodontitis is a complex mechanism, in which the presence of specific periodontal pathogens is necessary, but not sufficient, to fully explain the extent and severity of the observed periodontal destruction. Moreover, the destruction of periodontal support tissue is largely the effect of the imbalance in the patient immune response, triggered by periodontal pathogen-derived antigens and virulence factors. The immune response elicited by periodontal pathogenic bacteria includes mechanisms associated with both innate and adaptive responses, where the role of antimicrobial peptides and lipid mediators are related to these two arms of immunity, and have not been fully elucidated in relation to their mechanisms of action against periodontal pathogens. In this review, a discussion is presented on the characteristics of these molecules and their role in periodontal disease in relation to both protection and destruction of tooth supporting tissue during periodontal infection. The relevance of considering these mediators within the complex scenario of the immune response during periodontal diseases is also highlighted, since they are a fundamental part of the host immune response. Periodontal diseases should be analysed in a broader perspective, where the study of these types of molecules involved in the immune response of periodontal tissues, may help to develop new therapeutic approaches to periodontal diseases in the future.

Expression and clinical significance of defensin alpha 6 in colorectal cancer

PURPOSE: Defensin alpha 6 (DEFA6) is cationic short peptide with known functional activities in innate antimicrobial immunity. DEFA6 is also highly expressed in colorectal cancer tissue. The mechanism and function of DEFA6 have not been reported how to play a significant role in carcinogenesis and cancer progression. The aim of this study was to evaluate the protumorigenic functions of DEFA6 in the colorectal cancer cell line and the clinical significance of DEFA6 expression in colorectal cancer patients.METHODS: DEFA6 expression was investigated by immunohistochemistry in 151 cases of colorectal cancer tissue and the association of DEFA6 expression was correlated with patient's cancer charecteristics.RESULTS: Immunohistochemistry analysis showed that the DEFA6 protein was expressed higher in advanced cancer group (high T stage, patients with lymph node metastasis, patients with vascular invasion) than early cancer group (low T stage, patients without lymph node metastasis, patients without vascular invasion) (P=0.007/0.032/0.047).CONCLUSION: As patient's survival usually depends on migration and invasion of the cancer cell, the high expression of DEFA6 in colorectal cancer cell is associated with patient's cancer charecteristics and could be a biomarker for colorectal cancer. The future study about characterization of DEFA6 will clarify its specific role in oncogenesis and its therapeutic potential.

Fatores microbiológicos e imunológicos envolvidos no desenvolvimento da cárie precoce da infância / Microbiological and immunological factors for the development of early childhood caries

A cárie precoce da infância (CPI) é ainda um grave problema de saúde pública no mundo, principalmente em países em desenvolvimento. Estudos têm sugerido a associação da ingestão frequente de carboidratos fermentáveis como a sacarose, altas contagens de microrganismos cariogênicos e maior vulnerabilidade imunológica da criança na etiologia da CPI. O objetivo deste estudo foi avaliar os aspectos microbiológicos e imunológicos associados ao desenvolvimento da cárie precoce da infância. Crianças com idade entre 36 e 60 meses foram selecionadas e divididas em três grupos: LC - livres de cárie, CPI e CPI-S (CPI-severa). Questionário sobre os aspectos socioeconômico-culturais, hábitos de higiene bucal e diários de dieta foram respondidos pelos responsáveis. Foram coletadas amostras de saliva e biofilme dental das crianças e processadas para subsequentes avaliações laboratoriais. Em seguida, os níveis de IgA salivar total e contra GbpB de S. mutans foram determinados por ELISA e Western blot, respectivamente, as concentrações salivares dos peptídeos catiônicos antimicrobianos (PCAM): defensinas hBD-2 e hBD-3, catelicidina LL-37 e histatina 5 (HTN-5) por ELISA e a presença e os níveis salivares de Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. e Scardovia wiggsiae por qRT-PCR, sendo que estes dados foram correlacionados com os níveis salivares e no biofilme dental de estreptococos mutans (SM) e Lactobacillus spp. por meio de cultivo em meios específicos. Os resultados mostraram que as crianças com CPI-S apresentaram menor renda familiar quando comparadas às crianças LC ou CPI. Contudo, a ingestão de açúcar não diferiu entre os grupos. O grupo CPI-S apresentou maior contagem de SM na saliva/biofilme em relação aos grupos LC e CPI. Houve uma correlação positiva entre a resposta de IgA contra GbpB e os níveis de SM, quando a população geral foi avaliada. Quando apenas crianças com altos níveis de SM foram comparadas, o grupo CPI-S...

Early childhood caries (ECC) is still a serious public health problem worldwide, especially in developing countries. Studies have been suggested the association among frequent intake of fermentable carbohydrates such as sucrose, high cariogenic microorganism’s counts and child’s immune vulnerability in the etiology of ECC. The objective of this study was to evaluate the microbiological and immunological factors for the development of early childhood caries. 36 to 60 monthold children were selected and distributed into three groups: caries free (CF), ECC and S-ECC (severe-ECC). Questionnaires about socio-economic-cultural data, oral hygiene habits and food-frequency diary were completed by the parents. Saliva and dental biofilm were collected from children and processed for subsequent laboratorial tests. The following analyses were determined: total IgA and IgA response against S. mutans GbpB by ELISA and Western blot, respectively; salivary concentrations of antimicrobial peptides (AMPs): defensins hBD-2 and hBD-3, cathelicidin LL-37 and histatin 5 (HTN-5) by ELISA; salivary detection and quantification of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. and Scardovia wiggsiae by qRT-PCR, and these data were correlated with mutans streptococci (MS) and Lactobacillus spp. levels by culture in specific medium. Results showed that S-ECC children had reduced family income compared to ECC and CF. However, sugar intake did not differ among the groups. S-ECC group had higher MS count than CF/ECC groups. Positive correlations between salivary IgA response against GbpB and MS counts were found when the entire population was evaluated. When children with high mutans streptococci counts were compared, S-ECC group showed a significant decrease in IgA antibody levels against GbpB compared to CF group. This finding was not observed for ECC group. The present study showed positive correlations between salivary hBD-2 and HTN-5 with…

Fatores microbiológicos e imunológicos envolvidos no desenvolvimento da cárie precoce da infância / Microbiological and immunological factors for the development of early childhood caries

A cárie precoce da infância (CPI) é ainda um grave problema de saúde pública no mundo, principalmente em países em desenvolvimento. Estudos têm sugerido a associação da ingestão frequente de carboidratos fermentáveis como a sacarose, altas contagens de microrganismos cariogênicos e maior vulnerabilidade imunológica da criança na etiologia da CPI. O objetivo deste estudo foi avaliar os aspectos microbiológicos e imunológicos associados ao desenvolvimento da cárie precoce da infância. Crianças com idade entre 36 e 60 meses foram selecionadas e divididas em três grupos: LC - livres de cárie, CPI e CPI-S (CPI-severa). Questionário sobre os aspectos socioeconômico-culturais, hábitos de higiene bucal e diários de dieta foram respondidos pelos responsáveis. Foram coletadas amostras de saliva e biofilme dental das crianças e processadas para subsequentes avaliações laboratoriais. Em seguida, os níveis de IgA salivar total e contra GbpB de S. mutans foram determinados por ELISA e Western blot, respectivamente, as concentrações salivares dos peptídeos catiônicos antimicrobianos (PCAM): defensinas hBD-2 e hBD-3, catelicidina LL-37 e histatina 5 (HTN-5) por ELISA e a presença e os níveis salivares de Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. e Scardovia wiggsiae por qRT-PCR, sendo que estes dados foram correlacionados com os níveis salivares e no biofilme dental de estreptococos mutans (SM) e Lactobacillus spp. por meio de cultivo em meios específicos. Os resultados mostraram que as crianças com CPI-S apresentaram menor renda familiar quando comparadas às crianças LC ou CPI. Contudo, a ingestão de açúcar não diferiu entre os grupos. O grupo CPI-S apresentou maior contagem de SM na saliva/biofilme em relação aos grupos LC e CPI. Houve uma correlação positiva entre a resposta de IgA contra GbpB e os níveis de SM, quando a população geral foi avaliada. Quando apenas crianças com altos níveis de SM foram comparadas, o grupo CPI-S...

Early childhood caries (ECC) is still a serious public health problem worldwide, especially in developing countries. Studies have been suggested the association among frequent intake of fermentable carbohydrates such as sucrose, high cariogenic microorganism’s counts and child’s immune vulnerability in the etiology of ECC. The objective of this study was to evaluate the microbiological and immunological factors for the development of early childhood caries. 36 to 60 monthold children were selected and distributed into three groups: caries free (CF), ECC and S-ECC (severe-ECC). Questionnaires about socio-economic-cultural data, oral hygiene habits and food-frequency diary were completed by the parents. Saliva and dental biofilm were collected from children and processed for subsequent laboratorial tests. The following analyses were determined: total IgA and IgA response against S. mutans GbpB by ELISA and Western blot, respectively; salivary concentrations of antimicrobial peptides (AMPs): defensins hBD-2 and hBD-3, cathelicidin LL-37 and histatin 5 (HTN-5) by ELISA; salivary detection and quantification of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. and Scardovia wiggsiae by qRT-PCR, and these data were correlated with mutans streptococci (MS) and Lactobacillus spp. levels by culture in specific medium. Results showed that S-ECC children had reduced family income compared to ECC and CF. However, sugar intake did not differ among the groups. S-ECC group had higher MS count than CF/ECC groups. Positive correlations between salivary IgA response against GbpB and MS counts were found when the entire population was evaluated. When children with high mutans streptococci counts were compared, S-ECC group showed a significant decrease in IgA antibody levels against GbpB compared to CF group. This finding was not observed for ECC group. The present study showed positive correlations between salivary hBD-2 and HTN-5 with…

Origin of neurotoxins from defensins / 生理学报

There are at least three conserved protein folds shared by ion channel-targeted neurotoxins and antimicrobial defensins, including cysteine-stabilized α-helix and β-sheet fold (CSαβ), inhibitor cystine knot fold (ICK) and β-defensin fold (BDF). Based on a combined data of sequences, structures and functions, it has been proposed that these neurotoxins could originate from related ancient antimicrobial defensins by neofunctionalization. This provides an ideal system to study how a novel function emerged from a conserved structural scaffold during evolution. The elucidation of functional novelty of proteins not only has great significance in evolutionary biology but also will be helpful in guiding rational molecular design. This review describes recent progresses in origin of neurotoxins, focusing on the three conserved protein scaffolds.

Expression of intestinal defensin-5, soluble phospholipase A2 and lysozyme and the relation to bacterial translocation in rat models of acute liver failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the intestinal expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in acute liver failure (ALF) using rat models, and to determine the relation of these expressions to intestinal bacterial translocation.</p><p><b>METHODS</b>Forty-eight healthy male Sprague-Dawley rats were divided into a control group (n=8) and a model group (n=40; intraperitoneal injection of 10% D-galactosamine). The model group was further divided into five subgroups according to the time lapse after model establishment (8, 16, 24, 48, and 72 hours). At the end of the experiments, homogenates of mesenteric lymph nodes, liver and spleen were cultured in agar for bacterial outgrowth.Hematoxylin-eosin stained sections of liver and terminal ileum were examined under an optical microscope to assess pathological changes. mRNA expression of RD-5, sPLA2 and lysozyme in the terminal ileum was determined by reverse transcription-polymerase reaction (RT-PCR), and protein expression of sPLA2 and lysozyme from the same anatomic location was determined by western blotting and immunohistochemistry. Means between groups were compared with one-way analysis of variance.</p><p><b>RESULTS</b>ALF was successfully induced in the D-galactosamine injected rats. No bacteria grew in the organ cultures from the control group, while 8.3%, 37.5% and 58.3% of the rats in the 24-, 48-and 72-hour model groups showed positive cultures. Despite this, the structure of the terminal ileum from the rats in the 72-hour model group was nearly intact, without obvious necrosis of mucosal epithelial cells. Expression of RD-5 and sPLA2 mRNA in the model groups gradually increased at early time points and peaked at 16 hours after induction of ALF (1.291+/-0.153 and 1.131+/-0.128), which was significantly higher than that detected in the control group (0.725+/-0.116 and 0.722+/-0.112, t=69.25, 95.71, all P<0.01). After that, the expression of RD-5 and sPLA2 mRNA progressively decreased, and by 72 hours after the induction of ALF, the expression (0.415+/-0.104 and 0.425+/-0.076) was significantly lower than that of the control group (t=31.55 and 44.98, all P<0.01). Lysozyme mRNA expression in the model group peaked at 8 hours after ALF induction (1.211+/-0.107), which was higher than that of the control group at this time point (0.853+/-0.093), and by 72 hours after ALF induction it declined to 0.704+/-0.103, which was significantly lower than that of the control group (t=9.224; all P=0.009). In addition, at 72 hours after ALF induction the protein expression of both lysozyme and sPLA2 was significantly lower in the model group (0.327+/-0.086 and 0.382+/-0.057) than in the control group (0.583+/-0.121 and 0.650+/-0.093, t=12.28 and 15.83, P=0.004 and 0.001). Similar results were obtained with immunohistochemical staining.</p><p><b>CONCLUSION</b>The function of the ileal mucosal immune barrier in the rat model of acute liver failure decreased, along with decreases in expression of RD-5, sPLA2 and lysozyme in the Paneth cells.At the same time, the rate of organ bacterial translocation increased without obvious injury to the intestinal mucosa structure.</p>

Caracterização da resposta imunológica de Aedes aegypti e Culex quinquefasciatus à infecção pelo sorotipo 1 do Dengue vírus / Characterization of immune response of Aedes aegypti and Culex quinquefasciatus to infection by Dengue virus serotype 1

Os insetos podem atuar como pragas agrícolas e vetores de patógenos causadores de doenças ao homem e outros animais. Investigações a respeito do sistema imunológico de Ae. aegypti e Cx. quinquefasciatus poderão contribuir para o desenvolvimento de métodos de controle das doenças veiculadas por estes insetos, principalmente a dengue, enfermidade causadora de sério problema de saúde pública no mundo. Apesar de Ae. aegypti ser a única espécie vetora confirmada na transmissão do vírus Dengue no Brasil, considera-se também importante um melhor entendimento dos mecanismos imunológicos de Cx. quinquefasciatus tido como refratário ao vírus. Neste estudo foram utilizadas linhagens de Ae. aegypti e Cx. quinquefasciatus mantidas no Insetário do Departamento de Entomologia do CPqAM/FIOCRUZ. Três grupos experimentais de fêmeas com 10 dias de idade foram formados para cada espécie. Grupo I, composto por fêmeas alimentadas com solução sacarose (10 por cento); grupo II, fêmeas alimentadas com sangue limpo e grupo III, fêmeas alimentadas com sangue infectado com o sorotipo DENV-1. De cada grupo foram obtidos hemolinfa, glândula salivar, intestino médio e corpo gorduroso para avaliação da expressão dos antimicrobianos defensina e transferrina. Essa avaliação foi realizada através de PCR em Tempo Real utilizando o kit QuantiFast SYBR Green - One-Step qRT-PCR. A avaliação da hemodinâmica foi realizada utilizando 10 microlitros de hemolinfa de cada grupo, através da contagem das células em câmara de Neubauer...

Beta-Defensin 124 Is Required for Efficient Innate Immune Responses in Prostate Epithelial RWPE-1 Cells

PURPOSE: The present study aimed to determine the role played by beta-defensin 124 (DEFB124) in the innate immunity of prostate epithelial RWPE-1 cells during bacterial infection. MATERIALS AND METHODS: The expression of DEFB124 was examined by quantitative real-time polymerase chain reaction (PCR), Western blotting, and immunocytochemistry. Enzyme-linked immunosorbent assays and quantitative real-time PCR were performed to determine the production of cytokines and chemokines. Western blotting and chromatin immunoprecipitation studies were performed to assess the interaction between DEFB124 and nuclear factor-kappa B (NF-kappaB) in peptidoglycan (PGN)-stimulated RWPE-1 cells. By chemotaxis assay, we assessed the effect of DEFB124 on the migration of monocytes. RESULTS: Exposure to PGN induced DEFB124 upregulation and NF-kappaB activation through IkappaBalpha phosphorylation and IkappaBalpha degradation. Bay11-7082, an NF-kappaB inhibitor, blocked PGN-induced DEFB124 production. Also, NF-kappaB was shown to be a direct regulator and to directly bind to the -3.14 kb site of the DEFB124 promoter in PGN-treated human prostate epithelial RWPE-1 cells. When DEFB124 was overexpressed in RWPE-1 cells, interestingly, the production of cytokines (interleukin [IL] 6 and IL-12) and chemokines (CCL5, CCL22, and CXCL8) was significantly increased. These DEFB124-upregulated RWPE-1 cells markedly induced chemotactic activity for THP-1 monocytes. CONCLUSIONS: Taken together, these results provide strong evidence for the first time that increased DEFB124 expression via NF-kappaB activation in PGN-exposed RWPE-1 cells enhances the production of cytokines and chemokines, which may contribute to an efficient innate immune defense.

Expressions of differential proteins in the expressed prostatic secretion of patients with III A chronic prostatitis / 中华男科学杂志

<p><b>OBJECTIVE</b>To study the expressions of differential proteins in the expressed prostatic secretion (EPS) of patients with III A chronic prostatitis and healthy men.</p><p><b>METHODS</b>We collected EPS samples from 35 patients with III A chronic prostatitis and 18 age-matched healthy men, and detected the differentially expressed proteins in EPS by MALDI-TOF/MS. Based on the data obtained, we conducted a statistical analysis on the mass-to-charge (m/z) ratios of different proteins and a retrieval analysis on the relevant proteins using the protein database.</p><p><b>RESULTS</b>In the comparative studies of the III A chronic prostatitis patients and healthy men, 5 proteins were detected as at least 2-fold differentially expressed, which were probably brevinin-2Eg, big endothelin-1, alpha-defensin 15, beta-defensin 134 and prostatic steroid-binding protein C2. The m/z ratios were significantly up-regulated in 3 372, 3 487, 425 and 5 325 Da proteins (P < 0.01) and down-regulated in 10631Da (P < 0.01).</p><p><b>CONCLUSION</b>Proteins are differentially expressed in the EPS of III A chronic prostatitis patients and healthy men, and these proteins may be significantly correlated with the development and progression of III A chronic prostatitis.</p>

Infecção natural de Triatoma brasiliensis brasiliensis, Caicó, Rio Grande do Norte, pelo Trypanosoma cruzi e o estudo de peptídeos antimicrobianos em diferentes espécies de triatomíneos (Hemiptera, Heteroptera, Reduviidae) / Natural infection of Triatoma brasiliensis brasiliensis, Caicó, Rio Grande do Norte by trrypanosoma cruzi and the study of antimicrobial peptides in different triatomine species (hemiptera, heteroptera, reduviidae)

Triatoma brasiliensis é um dos vetores da doença de Chagas no Nordeste do Brasil, apresentando altos índices de infecção natural pelo Trypanosoma cruzi. No presente estudo, dois aspectos foram abordados: primeiro a coleta de exemplares de T. b. brasiliensis oriundos de Caicó, RN, para análise de infecção natural por T. cruzi, em períodos diferentes (abril e novembro de 2011). O segundo aspecto abordado está relacionado ao estudo do gene que codifica defensinas como marcador molecular filogenético para a análise de diferentes espécies de triatomíneos, incluindo o complexo T. brasiliensis, além de exemplares de T. b. brasiliensis coletados em Caicó. Em relação à infecção natural de T. b. brasiliensis por T. cruzi observamos um alto índice (86 porcento), mais especificamente na localidade de Penedo. Também foi observado que não houve diferenças significativas entre as expedições realizadas nos períodos de chuva (abril de 2011) e seca (novembro de 2011)Além disso, cinco isolados de T. cruzi foram caracterizados molecularmente por duas metodologias: mini-exon, 24Salpha rDNA e 18S rDNA. Como resultados observamos os dois genótipos (TcI e TcII) circulando em Caicó. Foram obtidas sequências do gene que codifica defensina, onde diferentes isoformas deste gene puderam ser identificadas e caracterizadas nas diferentes espécies de triatomíneos estudadas. A espécie Panstrongylus megistus ficou nos mesmos clados que representantes do gênero Rhodnius. Houve uma clara separação dos clados em relação aos gêneros Triatoma e Rhodnius. Com relação aos membros do complexo T. brasiliensis, não foi possível uma clara distinção entre as espécies pelo fato da molécula de defensina apresentar menos que 500 bp em seu tamanho, além de ser bastante conservada, não sendo portanto um bom marcador para separar grupos muito proximamente relacionados...

Triatoma brasiliensis is one of the main Chagas disease vectors in NortheasternBrazil, presenting a high natural Trypanosoma cruzi infection rate. In the present work, two different aspects were investigated: First, triatomines of the subspecies T. b. brasiliensis were collected in Caicó, RN for analysis of natural infection by T. cruzi in two dissimilar periods (April and November 2011). The second aspect is related touse of defensin encoding genes as a phylogenetic marker for the study of different triatomine species, mainly the T. brasiliensis species complex, including T. b. brasiliensis specimens from Caicó. The results showed a high infection rate (86 percent) of T. b. brasiliensis from Caicó by T. cruzi, especially in insects from Penedo. Nosignificant differences between the rainy period (April 2011) and dried season (November 2011) could be observed. In addition, five T. cruzi isolates were cultivated and characterized by three different molecular methods (mini-exon, 24Salpha rDNA and 18S rDNA). The results showed the presence of TcI and TcII genotypes circulating in Caicó. Defensin encoding genes were amplified and sequenced for different triatomine species, showing three differing forms. Panstrongylus megistus clusteredtogether with representatives of the genus Rhodnius. A separation between the genus Triatoma and Rhodnius could also be detected. However, inside of the T. brasiliensis species complex the taxa could not be well separated. Since thismolecular marker has a size of less than 500 bp and is highly conserved defensinencoding sequences might not be a good marker to separate closely related species and subspecies...

Antifungal indole and pyrrolidine-2,4-dione derivative peptidomimetic lead design based on in silico study of bioactive peptide families

The rise of opportunistic fungal infections highlights the need for development of new antimicrobial agents. Antimicrobial Peptides [AMPs] and Antifungal Peptides [AFPs] are among the agents with minimal resistance being developed against them, therefore they can be used as structural templates for design of new antimicrobial agents. In the present study four antifungal peptidomimetic structures named C[1] to C[4] were designed based on plant defensin of Pisum sativum. Minimum inhibitory concentrations [MICs] for these structures were determined against Aspergillus niger N402, Candida albicans ATCC 10231, and Saccharomyces cerevisiae PTCC 5052. C[1] and C[2] showed more potent antifungal activity against these fungal strains compared to C[3] and C[4]. The structure C[2] demonstrated a potent antifungal activity among them and could be used as a template for future study on antifungal peptidomemetics design. Sequences alignments led to identifying antifungal decapeptide [KTCENLADTY] named KTC-Y, which its MIC was determined on fungal protoplast showing 25 [micro g/ml] against Aspergillus fumigatus Af293. The present approach to reach the antifungal molecules seems to be a powerful approach in design of bioactive agents based on AMP mimetic identification

Vitamin D, the Cutaneous Barrier, Antimicrobial Peptides and Allergies: Is There a Link?

Atopic diseases such as atopic dermatitis (AD) are very common in industrialized countries. Up to 15%-30% of all children and 2%-10% of all adults suffer from AD. Already in early disease stages, a defective epidermal barrier is known to contribute to the pathogenesis of AD. Central elements in the epidermal barrier are antimicrobial peptides (AMPs), which are secreted by keratinocytes, sweat gland cells but also infiltrating immune cells. AMPs function as endogenous antibiotics and are able to kill bacteria, viruses, and fungi. Furthermore AMPs act as immune modulators with effects on the innate and adaptive immune system. The probably best studied AMPs in human skin are the defensins and cathelicidin. In atopic diseases the functions of AMPs such as cathelicidin might be impaired and microbial superinfections could serve as cofactors for allergic sensitization. Hence, induction of AMPs could be beneficial in these patients. Cathelicidin which is often referred to its peptide form hCAP18 or LL-37 can be induced by ultraviolet light B (UVB) irradiation and is upregulated in infected and injured skin. The cathelicidin gene carries a vitamin D response element and the vitamin D pathway could therefore be targeted for cathelicidin regulation. As the development and course of atopic diseases might be influenced by vitamin D signaling these pathomechanisms could explain the growing evidence connecting vitamin D to allergic diseases, including AD, allergic rhinitis, food allergies and asthma. In this review the role of vitamin D and the AMP cathelicidin in the pathogenesis of atopic diseases with impaired barrier function will be discussed.

The Expression of Human Beta Defensins by Thermal Injury

PURPOSE: Defensins are important components of innate immune system. These peptides have antimicrobial activity against a wise variety of pathogens that associated with burn wound infection. In particular, human beta-defensins are expressed in normal epidermal region and showed differential expression of some skin disease. We investigated that expression of human beta-defensin by in vitro and ex-vivo by thermal condition. METHODS: To investigate the expression of human beta-defensins in acute burn condition, we cultured keratinocytes and used to rat's skin at this experiment. After thermal condition, we showed the expression of beta-defensins-2 (hBD-2), -3 (hBD-3), keratins, keratinocyte differentiation and junction protein levels by RT-PCR and immunohistochemistry (IHC). RESULTS: HBD-2 & involucrin were down-regulated from 1 hr to 8 hrs in mRNA level. But others were not changed in mRNA level. In protein level, hBD-3 was decreased but pan-cytokeratin and beta-catenin were not changed. CONCLUSION: HBD-2 was down-regulated in thermal injury. Because thermal injury could induce the influence of keratinocyte differentiation and the decrease of skin protection ability. Our results suggested that human beta-defensins plays an important role in protection by several injury.

Mecanismos de resistencia natural al VIH en seres humanos: un resumen de 10 años de investigación en población colombiana: [revisión] / Mechanisms of human natural resistance to HIV: a summary of ten years of research in the Colombian population: [review]

La historia natural de la infección por el virus de la inmunodeficiencia humana de tipo 1 (VIH-1) es un proceso variable y complejo que, en forma similar a otras infecciones, ha hecho evidente la existencia de mecanismos de resistencia natural que pueden inhibir el establecimiento de la infección o su progresión hacia estadios avanzados. Cuando hay una exposición continua a partículas virales infecciosas, varios mecanismos genéticos e inmunitarios son esenciales para que se establezca la resistencia. El objetivo de este manuscrito fue revisar todos los mecanismos de resistencia al VIH-1, hasta el momento propuestos en seres humanos, y presentar los resultados más importantes que se han obtenido en diferentes estudios realizados en los últimos 10 años de investigación en esta área, en individuos colombianos, particularmente enfocados en determinar los mecanismos involucrados en la protección de un grupo de personas que se han expuesto repetidamente al virus, pero que han permanecido sin evidencia clínica ni serológica de infección por el VIH-1. Aunque los estudios llevados a cabo por nuestro grupo de investigación han corroborado el papel protector de algunos de los mecanismos de protección previamente propuestos, la investigación actual en esta área, a nivel mundial, ha hecho evidente que el fenómeno de resistencia natural depende de múltiples factores con una gran influencia genética, y que sólo mediante estudios multicéntricos que involucren individuos con diferente componente genético, podrán establecerse los mecanismos universales de protección. Profundizar en el conocimiento en esta área permitirá el desarrollo de nuevas medidas preventivas y terapéuticas para la infección por el VIH-1.

The natural history of human immunodeficiency virus type-1 (HIV-1) infection is a complex and variable process that, similarly to other infections, has clearly demonstrated the existence of mechanisms of human natural resistance. The resistance either inhibits the establishment of infection or delays disease progression. When there is continuous exposure to infectious viral particles, several genetic and immunological mechanisms are essential to lead to resistance to HIV-1 infection/progression. The objective of this manuscript was to review the different mechanisms so far proposed to be responsible for HIV-1 resistance and to present the main results derived from 10 years of research in this area among Colombian subjects. In particular, this review focuses on determining the mechanisms involved in the protection of a group of individuals repeatedly exposed to the virus but who remained exempt of serological and clinical evidence of HIV-1 infection. Although the studies carried out in our research group corroborated the protective role of some of the previously proposed mechanisms of protection, ongoing research worldwide has made it clear that the phenomenon of human natural resistance depends on multiple factors with an important genetic influence, and only multicenter studies involving individuals with different genetic backgrounds may determine more universal mechanisms of resistance. Increasing our knowledge in this field will contribute to the development of novel preventive and therapeutic measures.

Rotura prematura de membranas pré-termo e corioamnionite histológica: aspectos da resposta imune inata e de repercussões no período neonatal / Preterm premature rupture of membranes and histological chorioamnionitis: aspects of the innate immune response and of outcomes in the neonatal period

Introdução: A rotura prematura de membranas pré-termo (RPM-PT) é uma importante intercorrência obstétrica e a infecção da cavidade amniótica advinda do trato genital inferior é um dos principais fatores associados à sua fisiopatologia. As membranas corioamnióticas são barreiras mecânicas contra a ascensão de micro-organismos e possuem papel fundamental no sistema imune, pois são importantes fontes de mediadores inflamatórios como as citocinas e também de antimicrobianos naturais, como as defensinas. Em resposta à infecção, ocorrem recrutamento e ativação de leucócitos para as membranas fetais, o que caracteriza a corioamnionite histológica, que ativa a cascata inflamatória na interface materno-fetal e contribui com os mecanismos de enfraquecimento e rotura das membranas. Além acometer os tecidos gestacionais de gestações complicadas por RPM-PT, a corioamnionite histológica é um fator de risco para resultados adversos maternos e morbidades neonatais. Objetivos: 1) Quantificar a expressão de β defensinas (HBD1, 3 e 4) por membranas corioamnióticas de gestações complicadas por prematuridade associada à corioamnionite histológica; 2) Quantificar a expressão de RNA mensageiro (RNAm) e proteína de IL-18 em membranas corioamnióticas de mulheres com RPM-PT e correlacionar a expressão com a presença de corioamnionite histológica; 3) Avaliar os resultados neonatais adversos de gestações pré-termo complicadas por corioamnionite histológica...

Introduction: The preterm premature rupture of membranes (PPROM) is an important obstetric issue, and infection in the amniotic cavity from the lower genital tract is one of the main factors associated with its physiology. Chorioamniotic membranes are mechanical barriers against the microorganism’s ascension, and they play a fundamental role in the immune system, since they are important sources of inflammatory mediators, such as cytokines, and of natural antimicrobials, like as defensins. In response to infection, leukocytes are recruited and activated in fetal membranes, which characterizes histological chorioamnionitis. This condition activates the inflammatory cascade on the maternal-fetal interface and contributes to weakening mechanisms and membrane rupture. In addition to affecting the gestational tissues of pregnancies complicated by PPROM, histological chorioamnionitis is a risk factor for adverse maternal outcomes and neonatal morbidities. Objectives: 1) To quantify the expression of β defensins (HBD1, 3 and 4) by chorioamniotic membranes of pregnancies complicated by prematurity associated with histological chorioamnionitis; 2) To quantify the expression of IL-18 mRNA and protein in the chorioamniotic membranes of pregnant women with PPROM and correlate expression with histological chorioamnionitis; 3) To evaluate adverse neonatal outcomes in preterm pregnancies complicated by histological chorioamnionitis...

The Relaxing Effect of alpha-Defensin 1 on the Adrenergic Responses of Rat Bladder

Defensins, cysteine-rich cationic polypeptides released from neutrophils, are known to have powerful antimicrobial properties. In this study, we sacrificed 30 rats to investigate the effects of alpha-defensin 1 on detrusor muscle contractions in isolated rat bladder. From the experiments we found relaxing effects of alpha-defensin 1 on the contractions induced by phenylephrine (PE) but not by bethanechol (BCh) in the detrusor smooth muscles. To determine the mechanisms of the effects of alpha-defensin 1, the changes of effects on PE-induced contraction by alpha-defensin 1 pretreatment were observed after pretreatment of Rho kinase inhibitor (Y-27632), protein kinase C (PKC) inhibitor (Calphostin C), potent activator of PKC (PDBu; phorbol 12,13-dibutyrate), and NF-kappaB inhibitors (PDTC; pyrrolidinedithiocarbamate and sulfasalazine). The contractile responses of PE (10(-9) ~ 10(-4) M) were significantly decreased in some concentrations of alpha-defensin 1 (5x10(-9) and 5x10(-8) M). When strips were pretreated with NF-kappaB inhibitors (PDTC and sulfasalazine; 10(-7) ~ 10(-6) M), the relaxing responses by alpha-defensin 1 pretreatment were disappeared. The present study demonstrated that alpha-defensin 1 has relaxing effects on the contractions of rat detrusor muscles, through NF-kappaB pathway. Further studies in vivo are required to clarify whether alpha-defensin 1 might be clinically related with bladder dysfunction by inflammation process.