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INTRODUCTION: To study the expression of defensin-5 (RD-5), soluble phospholipase A2 (sPLA2) and lysozyme in the intestine in a rat model of acute liver failure and its relationship with intestinal bacterial translocation (BT). PATIENTS AND METHODS: Sprague-Dawley (SD) rats were divided into two groups. The experimental group was divided into five subgroups according to the lapsing time after the model was established, which were designated accordingly as 8h, 16h, 24h, 48h, and 72h groups. Acute liver failure (ALF) model was induced by intraperitoneal injection of 10% d-galactosamine. The homogenates of mesenteric lymph nodes (MLNs), liver and spleen from each group were cultured in agar to determine the bacterial outgrowth. The mRNA expression of RD-5, sPLA2, lysozyme and the protein expression of sPLA2, lysozyme were determined. RESULTS: No bacteria grew in the organ cultures from the control group while experimental groups had positive cultures. Expression of the RD-5 and sPLA2 mRNA in the experimental groups gradually increased at early time points and peaked 16h after induction of ALF, then progressively decreased. The mRNA expression of lysozyme in the experimental group peaked at 8h after ALF induction, then progressively decreased. Similar results were obtained with Western blot and immunohistochemical staining. DISCUSSION: The immune barrier function of the ileal mucosa in the rat model of acute liver failure was compromised as demonstrated by the decreased expression of RD-5, sPLA2 and lysozyme in Paneth cells along with increased intestinal bacterial translocation.

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BACKGROUND: The aim of this study was to assess the prognostic value of vitamin D, vitamin D binding protein (VDBP) and vitamin D-related peptides in septic shock patients in relation to hospital mortality. METHODS: This is a single-center, prospective, observational study that included all consecutive patients meeting criteria for septic shock who were admitted to the ICU. VDBP, 25-hydroxy vitamin D, 1,25-dihydroxy vitamin D, cathelicidin and beta-defensin levels were determined in blood samples obtained on admission to the ICU. RESULTS: Seventy-five patients were studied. The best area under the curve (AUC) for prediction of in-hospital mortality was for VDBP (0.78), with a negative predictive value of 85.45% for the optimal cut-off point. VDBP was also the only variable that had a statistically significant association with a higher risk of in-hospital mortality, regardless of other assessed variables and pre-determined confounders: adjusted odds ratio of 5.20 (95% confidence interval: 1.21-22.36). When restricted to patients with vitamin D insufficiency (n=54), the AUC was 0.77, and the adjusted OR 12.22 (95% CI: 1.46-102.14; p=0.021) for in-hospital mortality. CONCLUSIONS: VDBP levels showed a statistically significant association with in-hospital mortality, supporting the clinical utility of VDBP as a good prognostic marker in septic shock patients. Vitamin D and vitamin D-related peptides are not associated with in-hospital mortality. These results should be confirmed in a multicentre study with a larger sample size.

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Antimicrobial peptides (AMPs) are components of innate immune system, are present in all living organisms, acting quickly and in unspecific way as a chemical barrier and they have antimicrobial activity. β-defensins is a major family of AMPs with characteristic β- sheet-rich fold, six conserved Cys with particular spacing and intramolecular bonds. They are extensively studied in mammals but scarcely in snakes. The β-defensin Defb-Lm02, of Lachesis muta snake, is codified by a gene structured in three exons and has 38 amino acid residues, molecular weight of 4.6 kDa, net charge of +8 at pH 7 and isoelectric point (pI) of 10.35. The synthetic linear peptide of Defb-Lm02 was tested to antimicrobial activity: it presented the minimum inhibitory concentration against Gram negative bacteria: Escherichia coli 16 μg/mL and Citrobacter freundii 16 μg/mL; and Gram positive: Micrococcus luteus 4 μg/mL and Staphylococcus aureus 32 μg/mL. It did not inhibit the growth of Gram-negative Klebsiella pneumonia, Serratia marcescens, Providencia rettgeri and Morganella morganii until the concentration of 64 μg/mL. These results may be due to the linearity of synthetic peptide. As the defense tasks of β-defensin could be dependent on their 3D structure, the production of recombinant β-defensin may potentiate its antimicrobial activity or suggest a new function. The synthetic gene, maltose binding protein (MBP) – TEV protease recognition site – DefbLm02, was cloned into the pET28a expression vector. The protein was expressed in E. coli BL21 Star and C43, at two temperatures 30 ° C and 37 ° C. After 4 hours of induction with IPTG 1 mM, which showed the best condition of expression was BL21 Star at 30 °C. The major part of fusion protein was soluble after purification by affinity chromatography and after proteolysis. The protein fusion was checked by Western blot using an antibody anti-MBP.

Os peptídeos antimicrobianos (PAMs) fazem parte do sistema imune inato, presente em praticamente todas as formas de vida, age de maneira rápida e inespecífica, como uma barreira química e possuem atividade antimicrobiana. As β-defensinas são uma importante família de PAMs com características ricas em folhas β, seis Cys conservadas com espaçamento particular e ligações intramoleculares, são extensivamente estudadas em mamíferos, mas raramente em serpentes, e podem refletir uma adaptação de peptídeos de defesa epitelial do hospedeiro. A Defb-Lm02, uma defensina da serpente Lachesis muta, é codificada por um gene estruturado em três exons cuja tradução ao peptídeo maduro consiste em 38 resíduos de aminoácidos, massa molecular de 4,6 kDa, carga líquida de +8 em pH 7, ponto isoelétrico (pI) de 10,35. Esse peptídeo foi sintetizado na forma linear e testado para atividade antimicrobiana, resultando nas seguintes concentrações inibitórias mínima contra bactérias Gram negativa: Escherichia coli 16 μg/mL e Citrobacter freundii 16 μg/mL; contra Gram positiva: Micrococcus luteus 4 μg/mL e Staphylococcus aureus 32 μg/mL. Não inibiu o crescimento das bactérias Gram-negativas Klebsiella pneumonia, Serratia marcescens, Providencia rettgeri e Morganella morganii até a concentração de 64 μg/mL. Estes resultados podem ser devido à linearidade do peptídeo sintético. Sabemos que algumas atividades biológicas das β-defensinas são dependentes da sua estrutura 3D, portanto, a obtenção dessa toxina na forma recombinante pode potencializar sua atividade antimicrobiana ou até sugerir novas funções. O gene sintético, codificando a fusão proteína de ligação à maltose (MBP) - sítio de reconhecimento da protease TEV – DefbLm02, foi clonado no vetor de expressão pET28a. A proteína de fusão foi expressa em duas cepas de E. coli, BL21 Star e C43, em duas temperaturas, 30°C e 37°C. Após 4 horas de indução com IPTG 1 mM, a combinação BL21 Star e 30°C foi a que apresentou a melhor eficiência de expressão. A maior proporção da proteína foi obtida solúvel após purificação por cromatografia de afinidade, como também após a digestão com TEV. A proteína de fusão foi verificada por Western Blotting, utilizando anticorpo anti-MBP.

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Traditionally, calcitriol has been considered a calcium and phosphate regulating hormone, but has recently been shown to play a pivotal role in innate immunity. Many barrier and immune cells have membrane and intracellular receptors that recognize different microbial antigens. Activation of these receptors induces synthesis of 1α-hydroxylase, which acts on 25 hydroxyvitamin D to generate intracellular calcitriol. Calcitriol activates its receptor and enhances the synthesis of important human antibiotics like cathelicidin and ß2-defensin while inhibiting hepcidin. These pluripotent peptides have an important role in innate immunity, and their regulation is abnormal in hypovitaminosis D. The literature on their secretion mechanisms, levels in different organic fluids, mechanism of action, and relationship with vitamin D is reviewed here.

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OBJECTIVE: This study aimed to determine a possible correlation between oral mucosal disease and salivary concentrations of the antimicrobial peptides human beta-defensin-1 (hβD-1) and human betadefensin- 2 (hβD-2). METHOD: The present work focussed on the establishment of a reversed phase-high performance liquid chromatography (RP-HPLC) procedure to quantify human beta-defensins (hβD-1 and hβD-2) in saliva samples of patients with oral diseases such as lichen planus (n = 10), Behçet (n = 10) and recurrent apthous stomatitis (n = 10). RESULTS: Linear calibration range for hβD-1 and hβD-2 defensins was 1.67−200 µg mL-1 and 3.13− 100 µg mL-1 with R2 values of 0.9998 and 0.996, correspondingly. The concentration of beta-defensins in saliva was determined by comparing the peak areas of eluted hβD-1 and hβD-2 with that of their standards. The variation of the amount of beta-defensins was evaluated by comparisons of the results obtained from the patients with oral mucosal diseases before and after treatments and the control subjects. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 1.62 µg mL- 1 and 5.39 µg mL-1 for hβD-1 and 0.94 µg mL-1 and 3.13 µg mL-1 for hβD-2, respectively. CONCLUSION: The salivary beta-defensin concentration was significantly higher in patients with oral mucosal diseases than in healthy volunteers; furthermore, in patients with oral mucosal diseases, the concentration was significantly higher before treatment than after treatment.

OBJETIVO: Este estudio tuvo por objeto determinar una posible correlación entre la enfermedad de la mucosa oral y las concentraciones salivales de la beta-defensina humana 1 (hβD-1) y la beta-defensina humana 2 (hβD-2) de los péptidos antimicrobianos. MÉTODO: El presente trabajo estuvo encaminado al establecimiento de un procedimiento de cromatografía líquida de alta eficacia de fase reversa (RP-HPLC) para cuantificar las beta-defensinas humanas (hβD-1 y hβD-2) en muestras de saliva de pacientes con enfermedades orales como el liquen plano (n = 10), Behçet (n = 10), y la estomatitis aftosa recurrente (n = 10). RESULTADOS: El rango de calibración lineal de las defensinas hβD-1 y hβD-2 fue 1.67-200 µg mL-1 y 3.13-100 µg mL-1 con valores R2 de 0.9998 y 996, respectivamente. La concentración de beta-defensinas en la saliva se determinó utilizando el área de sus estándares. La variación de la cantidad de beta defensinas fue evaluada por comparaciones de los resultados obtenidos de los pacientes con enfermedades de la mucosa oral, antes y después de los tratamientos y los sujetos de control. Se halló que el límite de detección (LDD) y el límite de cuantificación (LDC) fueron 1.62 µg mL-1 y 5.39 µg mL- 1 para hβD-1 y 0.94 µg mL-1 y 3.13 µg mL-1 hβD-2, respectivamente. CONCLUSIÓN: La concentración de beta-defensina salival fue significativamente mayor en los pacientes con enfermedades de la mucosa oral que en los voluntarios sanos. Además, en pacientes con enfermedades de la mucosa oral, la concentración fue significativamente mayor antes del tratamiento que después del tratamiento.

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A presença de Candida albicans nos biofilmes microbianos da superfície interna das próteses totais superiores está relacionada com uma doença inflamatória no palato, a estomatite protética. Constituinte da defesa inata do hospedeiro, o epitélio bucal, por sua vez, tem a capacidade de reconhecer e reagir aos fatores fúngicos a fim de evitar a invasão pelo microrganismo. O objetivo deste trabalho foi avaliar in vitro o efeito direto e indireto de C. albicans viável sobre as células epiteliais de palato humano (CEPH) ao longo do tempo. Objetivamos correlacionar os eventos de agressão, apoptose e invasão das CEPH provocados pelo fungo, com as respostas de defesa epitelial mediante produção de óxido nítrico (NO) e expressão gênica do peptídeo antimicrobiano β-defensina 2 (hBD-2). Material e Métodos: As CEPH foram obtidas, parte pelo método explante e parte pelo método enzimático, e mantidas em co-cultivo sobre uma camada de sustentação feederlayer (fibroblastos gengivais humanos mitoticamente inativados). Após desafios das CEPH com C. albicans ATCC 90028 por contato direto fungo-epitélio (D.D.) e indireto pelo sobrenadante da cultura do fungo hifal (D.I.), proporções de desafio de 0,01/1; 0,025/1 e 0,1/1 levedura/queratinócito (FUN/EPI) e tempos experimentais de 3, 6 e 10 h foram determinados; via ensaios de viabilidade celular por imunofluorescência (LIVE/DEAD), e análise qualitativa da invasão celular pelo fungo por meio do método colorimétrico com laranja de acridina. A apoptose epitelial foi determinada pela marcação nuclear fluorescente com Hoechtst 33258. A produção de óxido nítrico (NO) e a expressão de RNAm de hBD-2 foram avaliados por reação colorimétrica de Griess e RT-qPCR, respectivamente. Os resultados foram expressos como média ± desvio padrão e submetidos aos testes estatísticos ANOVA Fatorial, Teste de Contraste; ou Teste de Mann-Whitney (p<0,05). Resultados: Em 3 h, foi detectado aumento da apoptose das células epiteliais em relação ao...

The presence of the fungus Candida albicans in the microbial biofilm underlying maxillary prosthesis is related to an inflammatory reaction of the palatal mucosa, the denture stomatitis. As a component of the host innate defense, the oral epithelium has the ability to recognize and react to fungal factors in order to prevent the microrganism invasion. The aim of this study was to in vitro evaluate the direct and indirect effect of viable C. albicans on the human palatal epithelial cells (HPEC) over time. The aggressive events, such as apoptosis and HPEC invasion by the fungus, were correlated with epithelial defense responses through the nitric oxide (NO) production and antimicrobial peptides β-defensin (hBD-2) mRNA expression. Methods: The HPEC were obtained by explant and enzymatic methods, and were maintained in co-culture on a feeder-layer support (mitotically inactivated human gingival fibroblasts). After the HPEC challenges with C. albicans ATCC 90028 by direct contact fungus-epithelium (D.D.) and indirect contact by supernatant from hyphal fungus (D.I.), defiance ratios of 0.01/1, 0.025/1 and 0.1/1 yeast/keratinocyte (FUN/EPI) and experimental times of 3, 6 and 10 h were determined. These conditions were standardized by cell viability immunofluorescence assay (LIVE/DEAD), and cell invasion qualitative analysis (colorimetric method with acridine orange). The apoptotic cells were determined by fluorescent nuclear staining with Hoechtst 33258. The nitric oxide (NO) production and hBD-2 gene expression were evaluated by Griess colorimetric reaction and RT-qPCR, respectively. The results were expressed as mean ± standard deviation and were analyzed using the factorial ANOVA, Contrast Test; or Mann-Whitney Test (p<0,05). Results: At 3 h, the apoptotic epithelial cells under 0.1/1 FUN/EPI increased compared to epithelium unchallenged (p<0,05) that remained over time with increasing concentration and independent of D.D. and D.I. The onset...

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Los inconvenientes del tratamiento con antibióticos, como el surgimiento de cepas multirresistentes y las reacciones adversas, han llevado a la búsqueda de alternativas. El conocimiento de las rutas de activación de la respuesta inmune innata y sus interacciones con las rutas de señalización de la respuesta inmune adaptativa podría llevar a tratamientos basados en elicitores de péptidos antimicrobianos, sustancias inocuas que producen la sobreexpresión de genes de la respuesta inmune innata, más efectivos, rápidos y seguros para combatir las infecciones, que siguen siendo un problema de salud pública mundial.

The drawbacks associated with antibiotic-based treatment of infectious diseases including an increase in multidrug-resistant strains and adverse reactions have lead to the search of antimicrobial peptide elicitors (APE), harmless substances that boost an overexpression of innate immunity genes. Knowledge on innate immunity activation pathways and their interactions with adaptive immunity would lead to more effective, faster and safer APE-based treatments to battle infections which still are a common public health problem worldwide.

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