Gut Inflammation Markers, Diet, and Risk of Islet Autoimmunity in Finnish Children - A Nested Case-Control Study.

BACKGROUND: Gut dysbiosis and increased intestinal permeability have been reported to precede type 1 diabetes-related autoimmunity. The role of gut inflammation in autoimmunity is not understood. OBJECTIVES: This study aimed to assess whether gut inflammation markers are associated with risk of islet autoimmunity and whether diet is associated with gut inflammation markers. METHODS: A nested case-control sample of 75 case children with islet autoimmunity and 88 control children was acquired from the Finnish Type 1 Diabetes Prediction and Prevention cohort. Diet was assessed with 3-d food records, and calprotectin and human ß-defensin-2 (HBD-2) were analyzed from stool samples at 6 and 12 mo of age. Conditional logistic regression analysis was used in a matched case-control setting to assess risk of autoimmunity. Analysis of variance, independent samples t test, and a general linear model were used in secondary analyses to test associations of background characteristics and dietary factors with inflammation markers. RESULTS: In unadjusted analyses, calprotectin was not associated with risk of islet autoimmunity, whereas HBD-2 in the middle (odds ratio [OR]: 3.23; 95% confidence interval [CI]: 1.03, 10.08) or highest tertile (OR: 3.02; 95% CI: 1.05, 8.69) in comparison to the lowest at 12 mo of age showed borderline association (P-trend = 0.063) with higher risk of islet autoimmunity. Excluding children with cow milk allergy in sensitivity analyses strengthened the association of HBD-2 with islet autoimmunity, whereas adjusting for dietary factors and maternal education weakened it. At age 12 mo, higher fat intake was associated with higher HBD-2 (ß: 0.219; 95% CI: 0.110, 0.328) and higher intake of dietary fiber (ß: -0.294; 95% CI: -0.510, -0.078), magnesium (ß: -0.036; 95% CI: -0.059, -0.014), and potassium (ß: -0.003; 95% CI: -0.005, -0.001) with lower HBD-2. CONCLUSIONS: Higher HBD-2 in infancy may be associated with higher risk of islet autoimmunity. Dietary factors play a role in gut inflammatory status.

Porcine ß-defensin-2 alleviates aflatoxin B1 induced intestinal mucosal damage via ROS-Erk1/2 signaling pathway.

Aflatoxin B1 (AFB1) is a highly toxic fungal toxin that causes severe damage to animal intestines. Porcine beta-defensin-2 (pBD-2) is a well-studied antimicrobial peptide in pigs that can protect animal intestines and improve productivity. This study aimed to investigate the molecular mechanisms of pBD-2 in alleviating AFB1-induced oxidative stress and intestinal mucosal damage using porcine intestinal epithelial cells (IPEC-J2 cells) and Kunming (KM) mice. The maximum destructive concentration of AFB1 for IPEC-J2 cells and the optimal therapeutic concentration of pBD-2 were determined by CCK-8 and RT-qPCR. We then investigated the oxidative stress and intestinal damage induced by AFB1 and the alleviating effect of pBD-2 by detecting changes of reactive oxygen species (ROS), inflammatory cytokines, tight junction proteins (TJPs) and mucin. Finally, the molecular mechanism of pBD-2 mitigates AFB1-induced oxidative stress and intestinal mucosal damage were explored by adding ROS and Erk1/2 pathway inhibitors to comparative analysis. In vivo, the therapeutic effect of pBD-2 on AFB1-induced intestinal damage was analyzed from aspects such as average daily gain (ADG), pathological damage, inflammation, and mucosal barrier in KM mice. The study found that low doses of pBD-2 promoted cell proliferation and prevented AFB1-induced cell death, and pBD-2 significantly restored the feed conversion rate and ADG of KM mice reduced by long-term exposed AFB1. Increasing the intracellular ROS and the expression and phosphorylation of Erk1/2, AFB1 promoted inflammation by altering inflammatory cytokines TNF-α, IL-1ß, IL-6, and IL-8, and disrupted the mucosal barrier by interfering with Claudin-3, Occludin, and MUC2, while pBD-2 significantly reduced ROS and decreased the expression and phosphorylation of Erk1/2 to restored their expression to alleviate AFB1-induced oxidative stress and intestinal mucosal damage in IPEC-J2 cells and the small intestine of mice.

Dissecting the relationship between antimicrobial peptides and mesenchymal stem cells.

Among the various biological properties presented by Mesenchymal Stem Cells (MSCs), their ability to control the immune response and fight pathogen infection through the production of antimicrobial peptides (AMPs) have been the subject of intense research in recent years. AMPs secreted by MSCs exhibit activity against a wide range of microorganisms, including bacteria, fungi, yeasts, and viruses. The main AMPs produced by these cells are hepcidin, cathelicidin LL-37, and ß-defensin-2. In addition to acting against pathogens, those AMPs have also been shown to interact with MSCs to modulate MSC proliferation, migration, and regeneration, indicating that such peptides exert a more diverse biological effect than initially thought. In the present review, we discuss the production of AMPs by MSCs, revise the multiple functions of these peptides, including their influence over MSCs, and present an overview of clinical situations in which the antimicrobial properties of MSCs may be explored for therapy. Finally, we discuss possibilities of combining MSCs and AMPs to generate improved therapeutic strategies.

Adsorption of Recombinant Human ß-Defensin 2 and Two Mutants on Mesoporous Silica Nanoparticles and Its Effect against Clavibacter michiganensis subsp. michiganensis.

Solanum lycopersicum L. is affected among other pests and diseases, by the actinomycete Clavibacter michiganensis subsp. michiganensis (Cmm), causing important economic losses worldwide. Antimicrobial peptides (AMPs) are amphipathic cationic oligopeptides with which the development of pathogenic microorganisms has been inhibited. Therefore, in this study, we evaluate antimicrobial activity of mesoporous silica nanoparticles (MSN5.4) loaded with human ß-defensin-2 (hßD2) and two mutants (TRX-hßD2-M and hßD2-M) against Cmm. hßD2, TRX-hßD2-M and hßD2-M presented a half-maximum inhibitory concentration (IC50) of 3.64, 1.56 and 6.17 µg/mL, respectively. MSNs had average particle sizes of 140 nm (SEM) and a tunable pore diameter of 4.8 up to 5.4 nm (BJH). AMPs were adsorbed more than 99% into MSN and a first release after 24 h was observed. The MSN loaded with the AMPs inhibited the growth of Cmm in solid and liquid media. It was also determined that MSNs protect AMPs from enzymatic degradation when the MSN/AMPs complexes were exposed to a pepsin treatment. An improved AMP performance was registered when it was adsorbed in the mesoporous matrix. The present study could expand the applications of MSNs loaded with AMPs as a biological control and provide new tools for the management of phytopathogenic microorganisms.

Cathelicidin levels in nasal secretions are associated with the severity of acute bronchiolitis.

OBJECTIVE: To investigate the association of serum vitamin D and nasal secretion antimicrobial peptides (AMPs) levels with the severity of acute bronchiolitis. STUDY DESIGN: We conducted a prospective single pediatric tertiary care center cohort study of inpatients aged 0-18 months with a first episode of acute bronchiolitis from November 1st 2014 to April 30th 2017. Disease severity was determined by the length of hospitalization and supplemental hospital data. Qualitative measurements included serum 25(OH)D and nasal secretion LL-37 and ß-defensin-2 levels. Correlations were examined with the Mann-Whitney and Kruskal-Wallis criteria for qualitative and the correlation coefficient Spearman's rho for quantitative factors. Multiple linear and logarithmic regression were performed to adjust for confounding factors. RESULTS: The study population consisted of 153 infants and toddlers with median age 3.1 months (interquartile range:1.6-4.9). No association was found between serum 25(OH)D and AMPs nasal secretions levels. Serum 25(OH)D and nasal secretion ß-defensin-2 levels were not associated with the severity of bronchiolitis. In contrast, LL-37 levels were inversely associated with the length of hospitalization (rho = -0.340, p = .001), the need for medication use (p = .001), as well as the duration of oxygen supplementation (rho = -0.339, p = .001), and intravenous fluid administration (rho = -0.323, p = .001). This association remained significant after adjustment for potential confounders. CONCLUSION: A significant association between LL-37 nasal secretions levels with the severity of acute bronchiolitis was found in hospitalized infants and toddlers. The role of LL-37 in the pathogenesis of bronchiolitis merits further investigation.

Human ß-defensin-2 suppresses key features of asthma in murine models of allergic airways disease.

BACKGROUND: Asthma is an airway inflammatory disease and a major health problem worldwide. Anti-inflammatory steroids and bronchodilators are the gold-standard therapy for asthma. However, they do not prevent the development of the disease, and critically, a subset of asthmatics are resistant to steroid therapy. OBJECTIVE: To elucidate the therapeutic potential of human ß-defensins (hBD), such as hBD2 mild to moderate and severe asthma. METHODS: We investigated the role of hBD2 in a steroid-sensitive, house dust mite-induced allergic airways disease (AAD) model and a steroid-insensitive model combining ovalbumin-induced AAD with C muridarum (Cmu) respiratory infection. RESULTS: In both models, we demonstrated that therapeutic intranasal application of hBD2 significantly reduced the influx of inflammatory cells into the bronchoalveolar lavage fluid. Furthermore, key type 2 asthma-related cytokines IL-9 and IL-13, as well as additional immunomodulating cytokines, were significantly decreased after administration of hBD2 in the steroid-sensitive model. The suppression of inflammation was associated with improvements in airway physiology and treatment also suppressed airway hyper-responsiveness (AHR) in terms of airway resistance and compliance to methacholine challenge. CONCLUSIONS AND CLINICAL RELEVANCE: These data indicate that hBD2 reduces the hallmark features and has potential as a new therapeutic agent in allergic and especially steroid-resistant asthma.

Effects of Acanthopanax senticosus supplementation on innate immunity and changes of related immune factors in healthy mice.

Modern scientific research has shown that Acanthopanax senticosus (AS) can regulate the innate immunity of healthy animals, thus affecting the health of animals. However, there are few systematic reports on the changes of innate immune indices of healthy animals after consuming AS. The purpose of this project was to study the effect on healthy mice's innate immunity and changes of related immune factors induced by feeding AS root powder supplementation. The results showed that the killing rate of natural cells increased in a dose-dependent manner in a certain time period. Compared to the control group, the treatment groups (T1, T2 and T3) improved significantly in the innate immune index (lysozyme, ß-defensin-2 and duodenal secretory IgA (SIgA) to varying degrees) and induced corresponding changes of immune factors at certain time periods. The correlation between SIgA and IFN-γ in mouse serum was enhanced, and the higher the concentration of AS in the diet, the stronger the correlation was. However, there was no significant difference in growth performance among groups. It is proved that AS supplementation can enhance innate immunity and change several relevant immune factors and cells of healthy mice without affecting growth performance.

Antibacterial and pro-osteogenic effects of ß-Defensin-2-loaded mesoporous bioglass.

The human antimicrobial peptide beta-defensin-2 (hBD2) shows broad antibacterial activity and infrequent bacterial resistance. Here mesoporous bioactive glass (MBG) was loaded with hBD2, forming hBD2-loaded MBG (BD-MBG). The antibacterial and osteogenic effects of BD-MBG were investigated in comparison with MBG and the blank control (BC). The result showed that BD-MBG yielded sustained hBD2 release for more than 7 weeks in vitro, and resulted in significantly lower amounts of viable bacteria and colony forming units, and significantly higher levels of bacterial protein release compared with those in the BC and MBG groups (all p<0.05). Compared with that in the BC group, significantly higher bone marrow stromal cell (BMSC) proliferation rates, alkaline phosphatase (ALP) activity, calcium nodule formation, and expression levels of early and late osteogenic makers were observed after MBG and BD-MBG treatments (p<0.05). Thus, BD-MBG inhibited bacterial growth, damaged their membrane, and promoted early and late osteogenic BMSC differentiation.

Effect of icariin on early ß-defensin-2 and T cell subsets in rats after tracheotomy. / ·ARTICLES··è®º 著·.

OBJECTIVES: To investigate the effect of icariin (ICA) on early ß-defensin-2 and T cell subsets in rats after tracheotomy. METHODS: A total of 54 SPF male Sprague-Dawley rats were randomly divided into a normal control group (group A), a model group (group B), and a model+ICA treatment group (group C), with 18 rats in each group. A tracheotomy intubation model of the B and C group was prepared. After 6 h of surgery, ICA intervention was given to group C. Groups A and B were given the same amount of normal saline. Lung tissue, alveolar lavage fluid and peripheral blood were taken at 24 h, 72 h and 168 h, respectively. The expression of rat ß-defensin-2 mRNA in lung tissue was detected by RT-PCR. The content of ß-defensin-2 in alveolar lavage fluid and peripheral blood serum was detected by ELISA. The content of peripheral blood T cell subsets (CD3+, CD4+, CD8+) was detected by flow cytometry, and the ratio of CD4+/CD8+ was calculated. RESULTS: After tracheotomy, the levels of ß-defensin-2 mRNA and ß-defensin-2 in lung tissue from the group B were increased significantly at 24 h, then they were decreased gradually, and decreased most significantly at 168 h (P<0.05). The content of ß-defensin-2 in peripheral blood of group B decreased gradually, and the content of ß-defensin-2 in 168 h was significantly lower than that in 24 h (P<0.05), but there was no significant difference between group B and group A (P>0.05). The level of CD3+ T cells in peripheral blood was significantly lower than that in the group A (P<0.05), but their was no significant difference in CD4+ and CD8+ T cells compared with group A (P>0.05). After ICA intervention in group C: lung tissue, alveolar lavage fluid, peripheral blood serum ß-defensin-2 content, and peripheral blood CD3+ and CD4+ T cell levels were gradually increased, significantly higher than those in the group B (P<0.05). CD8+ T cell level was significantly lower than that in the group A at 24 h (P<0.05), the CD4+/CD8+ ratio was significantly higher at 168 h than those in the group A or B (both P<0.01). CONCLUSIONS: ICA can improve the early lung immune function in rats with tracheotomy, which might be related to up-regulation of ß-defensin-2 in lung tissue and alveolar lavage fluid, concomitant with increases in CD3+ and CD4+ T cells and CD4+/CD8+ ratio in peripheral blood while reduction in CD8+ cells.

Lung allograft donors with excessive alcohol use have increased levels of human antimicrobial peptide LL-37.

The relatively low long-term survival rate of lung transplant recipients as compared to other organ recipients serves as an impetus to identify potential lung dysfunction as early as possible. There is an association between donor heavy alcohol use and acute lung injury in the lung allograft after transplant, known as primary graft dysfunction. Excessive alcohol use (EAU) can induce pulmonary immune dysregulation in response to an infection. Antimicrobial peptides (AMPs) are an important component of the innate immune response to pulmonary infections, but the impact of EAU on AMPs in the allograft lung has not been evaluated. Our hypothesis is that specific lung AMPs, LL-37, α-defensin-1,2,3, and ß-defensin-2, are dysregulated in the lungs from organ donors who had EAU. In this prospective observational investigation, we measured AMPs via ELISA and inflammatory cytokines via multiplex bead array, in bronchoalveolar lavage (BAL) fluid of lung allograft donors, comparing results based on their alcohol consumption. LL-37 levels in lung donors with EAU were found to be increased compared to nondrinker (ND) donors [median 7.7 ng/mL (IQR 4.1-37.0) vs. 2.3 ng/mL (IQR 1.1-7.9), p = 0.004], whereas α-defensins-1,2,3 were decreased only in the presence of an infection in donors with EAU compared to ND donors [median 2.2 ng/mL (IQR 1.6-2.4) vs. 3.2 ng/mL (IQR 2.3-3.8), p = 0.049]. There was no difference in ß-defensin-2 levels. Gene expression levels of these AMPs were not different. Elevated levels of CXCL8 were noted in bronchial washings of donors with EAU compared to ND donors, [median 4372 pg/mL (IQR 3352-13180) vs. 867.3 pg/mL (IQR 163.6-3675), p = 0.04], suggesting a potentially heightened inflammatory response. At 1 month post-transplant, LL-37 and CXCL8 levels are decreased compared to levels at time of transplant. In lung donors with EAU, LL-37 and α-defensins-1,2,3 dysregulated levels in the presence of an infection may be a harbinger of dysfunction of the lungs through the transplant process.

ATP serves an anti-inflammatory role by enhancing ß-defensin-2 response in acute pneumonia of rat.

The aim of the current study was to evaluate the effect of ATP on the expression of rat ß-defensin-2 (rBD-2) in a time-dependent manner, as well as its therapeutic value in an acute pneumonia rat model. A total of 30 rats as a treatment group and 30 as a control group were treated with the same dose of ATP and normal saline, respectively, lung tissues were isolated from rat and expression of rBD-2 mRNA was assessed with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) at 12, 24 and 36 h following treatment. Rats were divided into five groups: The control group treated with normal saline, the Pseudomonas aeruginosa (PA) infected group, group treated with ATP, group treated with cephalosporins, and the group treated with both ATP and cephalosporins. At 24 h following treatment, rat serum and lung tissues were collected for assessment of histological changes, and alterations to expression of the rBD-2 protein by immunohistochemistry, expression of tumor necrosis factor (TNF)-α and interleukin (IL)-6 proteins by ELISA. RT-qPCR results indicated that the expression of rBD-2 mRNA was upregulated in response to ATP stimulation in lung tissues of rat, reaching its highest peak at 24 h. Immunohistochemistry demonstrated that ATP treatment enhanced the expression of rBD-2 protein in rat lungs. Ceftazidime and ATP protected lungs from infection of PA and reduced the pathological damage of the lung. Overexpression of rBD-2 by ATP led to decreased protein expression of TNF-α and IL-6 in lung tissues and serum. ATP upregulates the expression of rBD-2 and serves an anti-inflammatory role in the acute pneumonia of a rat model.

Human ß-defensin-2 production from S. cerevisiae using the repressible MET17 promoter.

BACKGROUND: Baker's yeast Saccharomyces cerevisiae is a proven host for the commercial production of recombinant biopharmaceutical proteins. For the manufacture of heterologous proteins with activities deleterious to the host it can be desirable to minimise production during the growth phase and induce production late in the exponential phase. Protein expression by regulated promoter systems offers the possibility of improving productivity in this way by separating the recombinant protein production phase from the yeast growth phase. Commonly used inducible promoters do not always offer convenient solutions for industrial scale biopharmaceutical production with engineered yeast systems. RESULTS: Here we show improved secretion of the antimicrobial protein, human ß-defensin-2, (hBD2), using the S. cerevisiae MET17 promoter by repressing expression during the growth phase. In shake flask culture, a higher final concentration of human ß-defensin-2 was obtained using the repressible MET17 promoter system than when using the strong constitutive promoter from proteinase B (PRB1) in a yeast strain developed for high-level commercial production of recombinant proteins. Furthermore, this was achieved in under half the time using the MET17 promoter compared to the PRB1 promoter. Cell density, plasmid copy-number, transcript level and protein concentration in the culture supernatant were used to study the effects of different initial methionine concentrations in the culture media for the production of human ß-defensin-2 secreted from S. cerevisiae. CONCLUSIONS: The repressible S. cerevisiae MET17 promoter was more efficient than a strong constitutive promoter for the production of human ß-defensin-2 from S. cerevisiae in small-scale culture and offers advantages for the commercial production of this and other heterologous proteins which are deleterious to the host organism. Furthermore, the MET17 promoter activity can be modulated by methionine alone, which has a safety profile applicable to biopharmaceutical manufacturing.

Construction of Bacillus subtilis strain engineered for expression of porcine ß-defensin-2/cecropin P1 fusion antimicrobial peptides and its growth-promoting effect and antimicrobial activity.

OBJECTIVE: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ß-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. METHODS: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. RESULTS: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. CONCLUSION: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.

Vitamin D nutritional status and vitamin D regulated antimicrobial peptides in serum and pleural fluid of patients with infectious and noninfectious pleural effusions.

BACKGROUND: Vitamin D and vitamin D dependent antimicrobial peptides such as Cathelicidin (LL-37) and ß-defensin 2 have an important role in innate and adaptative immunity, but their role in pleural effusions has not been studied before. METHODS: Serum and pleural fluid samples from 152 patients with pleural effusion were collected, corresponding to 45 transudates and 107 exudates, 51 infectious effusions (14 complicated and 37 non-complicated), 44 congestive heart failure effusions and 38 malignant effusions. The levels of 25 OH-vitamin D, 1,25-(OH)2-vitamin D, Vitamin D Binding Protein (VDBP), LL-37 and ß-defensin 2, both in serum and pleural fluid were evaluated in this prospective study. Differences between groups were analysed using unpaired t tests or Mann-Whitney tests. Correlations between data sets were examined using Pearson correlation coefficient or Spearman rank correlation coefficient. Diagnostic accuracy was estimated using ROC curve analysis. RESULTS: Low serum 25 OH vitamin D levels were found in all groups. Infectious effusions (IE) had higher serum and pleural fluid LL-37 levels compared to congestive heart failure or malignant effusions. Among IE, complicated had higher serum and pleural fluid LL-37 levels, and lower serum ß-defensin-2 levels. Positive correlations were found between serum 25 OH-vitamin D levels and serum or pleural 1,25-(OH)2-vitamin D levels, and between 1,25-(OH)2-vitamin D and LL-37 serum. Diagnostic accuracy of the different molecules was moderate at best. CONCLUSIONS: Hypovitaminosis D is highly prevalent in pleural effusions. LL-37 is produced intrapleurally in IE. This production is higher in complicated IE. No evidence of pleural production of ß-defensin 2 was found in any of the groups. Diagnostic accuracy of the different molecules is at the best moderate for discriminating different types of effusions.

Oral administration of synthetic porcine beta-defensin-2 improves growth performance and cecal microbial flora and down-regulates the expression of intestinal toll-like receptor-4 and inflammatory cytokines in weaned piglets challenged with enterotoxigenic Escherichia coli.

Synthetic porcine beta-defensin-2 (pBD-2) was tested as an alternative to antimicrobial growth-promoters in pig production. Thirty 21-day weaned piglets were challenged with enterotoxigenic Escherichia coli, and orally dosed with either sterile water (CON), pBD-2 (BD) or neomycin sulphate (NS) twice daily for 21 days. pBD-2 and NS led to higher growth performance, jejunum villus height and increased expression of insulin-like growth factor-I compared with the CON group (P < 0.05). Hemolytic E. coli scores from rectal swabs, and copy numbers of E. coli, Bacteroides fragilis and Streptococcus in the cecal digesta of the BD- or NS-treated piglets were lower than those in the CON group (P < 0.05). Messenger RNA levels of toll-like receptor 4, tumor necrosis factor-α, interleukin (IL)-1ß, and IL-8 in the jejunum mucosa of the BD and NS groups were lower than those in the CON group (P < 0.05). Copy numbers of Lactobacilli and Bifidobacteria in the cecal digesta of the BD group were higher than those of the CON and NS groups (P < 0.05). Therefore, pBD-2 has antimicrobial activity in piglets, and it can improve growth performance, reduce inflammatory cytokine expression and affect intestinal morphological indices in the same way as probiotics. © 2015 Japanese Society of Animal Science.

Murine ß-defensin-2 may regulate the effect of bacillus Calmette-Guérin (BCG) in normal mouse bladder.

PURPOSE: We investigated whether bacillus Calmette-Guérin (BCG)-induced secretion of murine ß-defensin-2 (mBD2) and determined whether mBD2 regulated BCG effects in the normal mouse bladder. MATERIALS AND METHODS: A total of 140 C57BL/6 female mice were divided into 28 groups, and the experiment was performed over 3 steps. In the first step (20 groups), mice bladders were stimulated with different doses of BCG (multiplicity of infection [MOI] 0, 1, 10, 30, and 100) and histological analysis was conducted in bladder specimens isolated at different times (0, 4, 8, and 24h after instillation) to determine optimal dose and time point of BCG internalization and urine mBD2 and cytokine concentration. In the second step (4 groups), BCG internalization and urine cytokine levels were measured after pretreatment of different recombinant mBD2 (rmBD2) (0, 1, 2.5, and 5 ng/ml) at optimal dose and time point. In the third step (4 groups), BCG internalization and urine cytokine levels were compared between pretreatment conditions (control, rmBD2, anti-mBD2 Ab, and rmBD2+anti-mBD2 Ab). Urine was collected for estimating mBD2 levels and a multiplex analysis for 9 cytokines. Real-time polymerase chain reaction assay was used for estimating the relative BCG cell number in mice bladder tissue. RESULTS: Bladder edema was induced by BCG (MOI 30 and 100), which progressed to an inflammatory infiltrate composed primarily of neutrophils and increased mBD2 secretion at 4 hours after instillation. Relative BCG cell number and urinary cytokine levels (interferon-γ and interleukins [IL]-2, -4, -6, and -10) response pattern was characterized by a peak at 4 hours after instillation followed by rapid decline. The levels of interferon-γ, and IL-1ß, -2, -4, -6, and -10 and relative BCG cell numbers decreased in a dose-dependent manner according to pretreatment with rmBD2 protein, and the responses were potentiated in the anti-mBD2 pretreatment group at 4 hours after BCG (MOI 30) instillation. CONCLUSION: The present results suggest that the mouse urothelium produces mBD2 in response to intravesicular BCG as a defense mechanism against BCG, and blocking mBD2 by an anti-mBD2 antibody increased the effectiveness of BCG.

Effects of recombinant IL-17F intranasal inoculation against Streptococcus pneumoniae infection in a murine model.

Interleukin-17F (IL-17F) is an important member of IL-17 cytokine family, which plays important roles in host defense against microbial infections. Streptococcus pneumoniae is a common pathogen associated with several invasive and noninvasive pneumococcal diseases, and mucosal immune response plays crucial roles in defenses against pneumococcal infection. Thus, intranasal inoculation may be an alternative approach against pneumococci. In this study, BALB/c mice were intranasally inoculated with recombinant IL-17F (rIL-17F) prior to S. pneumoniae (American Type Culture Collection 6303, serotype 3) infection. As compared with the control group, numbers of total leukocyte, neutrophil, and macrophage in lungs were significantly increased in mice inoculated with rIL-17F. The levels of macrophage inflammatory protein 1α (MIP-1α), MIP-2ß, and interferon γ were significantly increased in bronchoalveolar lavage fluid and culture supernatant of splenocytes from mice inoculated with rIL-17F. rIL-17F inoculation also significantly elevated ß-defensin-2 expression in lung tissues. Furthermore, compared with S. pneumoniae infection group, rIL-17F inoculation prior to infection significantly reduced S. pneumoniae colonization in lungs. These findings demonstrated that rIL-17F intranasal inoculation strengthened host defense against pneumococci, which may be developed to prevent pneumococcal infection.

Periodontopathogens and human ß-defensin-2 expression in gingival crevicular fluid from patients with periodontal disease in Guangxi, China.

BACKGROUND AND OBJECTIVE: Periodontal diseases are often induced by periodontopathogens, which are always exposed to certain innate immune factors in gingival crevicular fluid, including human ß-defensin-2 (hBD-2). This study aims to investigate the relationship among periodontopathogens, clinical parameters and hBD-2 expression. MATERIAL AND METHODS: Thirty-two healthy controls, 42 patients with chronic gingivitis and 95 patients with chronic periodontitis were recruited in Guangxi, China. Bleeding index, probing depth and clinical attachment level were measured for all teeth including mesiobuccal, buccal, disobuccal, mesiolingual, lingual, disolingual six sites of all patient. Gingival crevicular fluid samples were collected from the study sites. The prevalence and copy numbers (CN) of Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola, Tannerella forsythia and total bacteria in gingival crevicular fluid were quantified by real-time PCR. The hBD-2 concentration in gingival crevicular fluid was measured by ELISA. RESULTS: Both the prevalence and the CN of A. actinomycetemcomitans, P. gingivalis, T. denticola and T. forsythia were higher in patients with chronic periodontitis than in healthy controls and patients with chronic gingivitis; however, there was no significant difference in the prevalence of P. intermedia among the three study groups, and the highest CN was found in patients with chronic gingivitis, rather than in patients with chronic periodontitis. The loads of P. gingivalis, P. intermedia, T. denticola and total bacteria were positively related to probing depth, bleeding index and clinical attachment level. The concentration of hBD-2 in gingival crevicular fluid was higher in patients with chronic gingivitis and in patients with chronic periodontitis than in healthy controls. In addition, the hBD-2 concentration was positively related to the CN of P. gingivalis, T. forsythia and total bacteria, as well as to bleeding index and probing depth. CONCLUSION: The prevalence, composition and CN of periodontopathogens were closely related to the severity of periodontal disease, and the red complex was related to the severity of clinical symptoms of periodontal diseases. The concentration of hBD-2 in gingival crevicular fluid from periodontal disease sites was higher than that in gingival crevicular fluid from healthy sites, which suggests that hBD-2 expression might be up-regulated by periodontopathogens.

A study on ß-defensin-2 and histatin-5 as a diagnostic marker of early childhood caries progression

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.