Potential role of salivary vitamin D antimicrobial peptide LL-37 and interleukins in severity of dental caries: an exvivo study.

INTRODUCTION: Vitamin D performs various functions as a hormone by promoting calcium absorption but plays a major role in innate immunity,cell differentiation, cell maturation through its genomic effects via vitamin D receptor. The immune response also plays a major role in tooth surface and supporting structure destruction and playing a major factor in high caries formation. The inflammatory cytokines are released has proinflammatory cytokines and stimulate cells in disease process. Therefore, in the present study we have evaluated the association of salivary vitamin D, LL-37, interleukins 6 and 17A in various levels of severity of dental caries. METHOD: Ethical approval was obtained (NU/CEC/2020/0339), 377 individuals reporting to department of conservative dentistry and endodontics, AB Shetty memorial institute of dental sciences were included based on inclusion criteria. The individuals were further divided into caries free(N = 105) and caries active(N = 272) based on their caries prevalence. The salivary were collected and evaluated for vitamin D, LL-37,IL-17A and IL-6.Results were statistically analysed with SPSS vs 22 (IBM Corp, USA). Normally distributed data were expressed as mean ± SD. Skewed data were expressed as median and interquartile range. To compare (mean) outcome measures between the two groups unpaired independent t-test was applied and for values in median IQR, Mann Whitney U test was used. All statistical analysis for P value were two-sided and significance was set to P ≤ 0.05. RESULTS: The study showed that, the salivary vitamin D statistically decreased with increasing severity of caries which showed that vitamin D plays an important role in prevention of caries. Antimicrobial peptide LL-37 was higher in caries free group but was not statistically significant, salivary IL-6 level was higher in caries active group but intergroup comparison did not show significant difference. Salivary IL-17A did not show statistically significant between caries active and caries free group. CONCLUSION: The salivary levels of vitamin D may play a vital role in prevalence of dental caries and its severity which can be a underlying cause in presence of other etiological factors.

Evaluation of biocompatibility and angiogenic potential of extracellular matrix hydrogel biofunctionalized with the LL-37 peptide.

BACKGROUND: Biomaterials must allow revascularization for a successful tissue regeneration process. Biomaterials formulated from the extracellular matrix (ECM) have gained popularity in tissue engineering because of their superior biocompatibility, and due to their rheological properties, ECM-hydrogels can be easily applied in damaged areas, allowing cell colonization and integration into the host tissue. Porcine urinary bladder ECM (pUBM) retains functional signaling and structural proteins, being an excellent option in regenerative medicine. Even some small molecules, such as the antimicrobial cathelicidin-derived LL-37 peptide have proven angiogenic properties. OBJECTIVE: The objective of this study was to evaluate the biocompatibility and angiogenic potential of an ECM-hydrogel derived from the porcine urinary bladder (pUBMh) biofunctionalized with the LL-37 peptide (pUBMh/LL37). METHODS: Macrophages, fibroblasts, and adipose tissue-derived mesenchymal stem cells (AD-MSC) were exposed pUBMh/LL37, and the effect on cell proliferation was evaluated by MTT assay, cytotoxicity by quantification of lactate dehydrogenase release and the Live/Dead Cell Imaging assays. Moreover, macrophage production of IL-6, IL-10, IL-12p70, MCP-1, INF-γ, and TNF-α cytokines was quantified using a bead-based cytometric array. pUBMh/LL37 was implanted directly by dorsal subcutaneous injection in Wistar rats for 24 h to evaluate biocompatibility, and pUBMh/LL37-loaded angioreactors were implanted for 21 days for evaluation of angiogenesis. RESULTS: We found that pUBMh/LL37 did not affect cell proliferation and is cytocompatible to all tested cell lines but induces the production of TNF-α and MCP-1 in macrophages. In vivo, this ECM-hydrogel induces fibroblast-like cell recruitment within the material, without tissue damage or inflammation at 48 h. Interestingly, tissue remodeling with vasculature inside angioreactors was seen at 21 days. CONCLUSIONS: Our results showed that pUBMh/LL37 is cytologically compatible, and induces angiogenesis in vivo, showing potential for tissue regeneration therapies.

Structural differences of neutrophil extracellular traps induced by biochemical and microbiologic stimuli under healthy and autoimmune milieus.

Neutrophil extracellular traps (NETs) are networks of decondensed chromatin loaded with antimicrobial peptides and enzymes produced against microorganisms or biochemical stimuli. Since their discovery, numerous studies made separately have revealed multiple triggers that induce similar NET morphologies allowing to classify them as lytic or non-lytic. However, the variability in NET composition depending on the inducer agent and the local milieu under similar conditions has been scarcely studied. In this work, a comparative study was conducted to evaluate structural and enzymatic divergences in NET composition induced by biochemical (phorbol myristate acetate [PMA] and hypochlorous acid [HOCl]) and microbiologic (Candida albicans, Staphylococcus aureus, and Pseudomonas aeruginosa) stimuli, along with the presence of plasma from healthy donors or patients with systemic lupus erythematosus (SLE). The results showed a differential composition of DNA and the antimicrobial peptide cathelicidin (LL37) and a variable enzymatic activity (neutrophil elastase, cathepsin G, myeloperoxidase) induced by the different stimuli despite showing morphologically similar NETs. Additionally, SLE plasma´s presence increased DNA and LL37 release during NET induction independently of the trigger stimulus but with no enzymatic activity differences. This work provides new evidence about NET composition variability depending on the inducer stimulus and the local milieu.

Can Salivary Biomarkers Be Used as Predictors of Dental Caries in Young Adolescents?

BACKGROUND Identifying caries predictors in the subpopulation at risk is one of the preconditions for developing effective caries prevention measures. The present exploratory study aimed to examine the significance of socio-demographic characteristics, dietary-hygiene habits, salivary pH, and salivary antimicrobial HNP-1, hBD-2, and LL-37 peptides as potential caries risk predictors in children ages 11-13 years. MATERIAL AND METHODS This prospective 1-year study enrolled 213 children ages 11-13 years. The subjects underwent a dental examination and their mothers were interviewed. Unstimulated saliva was collected from the subjects to determine its pH value, as well as the salivary levels of HNP-1, hBD-2, and LL-37 peptides in 85 of the subjects. After 12 months, the 1-year caries incidence rate was recorded. Logistic regression analysis was used to estimate the ability of selected variables to predict caries risk. RESULTS The univariable logistic regression analysis determined that the most significant independent caries risk predictors were: sex (female) (OR=2.132, p=0.007), mothers' education (OR=1.986, p=0.020), salivary pH (OR=0.270, p=0.043), oral hygiene index (OR=1.886, p=0.015), and daily tooth brushing frequency (OR=0.565, p=0.042). The multivariable model showed that sex and oral hygiene-related variables were the most important caries predictors. CONCLUSIONS Salivary HNP-1, hBD-2, and LL-37 peptides were not found to have a significant predictive value. Therefore, socio-demographic and oral hygiene variables remain important caries predictors in early adolescents, suggesting the importance of the mechanical control of biofilm as the key measure for preventing caries. However, there is still a need for effective caries risk biomarkers, and additional research is needed in this area of caries risk prediction.

Studies on citrullinated LL-37: detection in human airways, antibacterial effects and biophysical properties.

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.

The impact of cathelicidin, the human antimicrobial peptide LL-37 in urinary tract infections.

BACKGROUND: The defense mechanisms of the urinary tract are attributed mainly to the innate immune system and the urinary tract urothelium which represent the first line of defense against invading pathogens and maintaining sterility of the urinary tract. There are only a few publications regarding cathelicidin (LL-37) and a urinary tract infection (UTI). This study was done to investigate the plasma and urine levels of human LL-37 in patients with UTI. METHODS: A case-control study was conducted at Omdurman Hospital, Sudan during the period from August 2014 to May 2017. The cases were patients with confirmed UTI and the controls were healthy volunteers without UTI. Sociodemographic and clinical data were obtained from each participant using questionnaires. Urine cultures and antimicrobial susceptibility were tested. Plasma and urine levels of LL-37 were determined using an enzyme-linked immunosorbent assay (ELISA) kit. SPSS (version 16.0) was used for analyses. RESULTS: Cases and controls (87 in each arm) were matched according to their basic characteristics. Compared with controls, the median (inter-quartile) LL-37 level in plasma [2.100 (1.700-2.700) vs. 1.800 (1.000-2.200) ng/ml, P = 0.002] and in urine [0.900 (0.300-1.600) vs. 0.000 (0.000-1.000) ng/mg creatinine, P < 0.001] was significantly higher in cases. There was no significant difference in the median plasma [2.1 (1.7-2.9) vs. 2.000 (1.700-2.400) ng/ml, P = 0.561] and urine [0.850 (0.275-2.025) vs. 0.900 (0.250-1.350) ng/mg creatinine, P = 0.124]. The uropathogenic Escherichia coli (UPEC) was the predominant isolate, n = 38 (43.7%). LL-37 levels between the E. coli isolates and the other isolated organisms. There was no significant correlation between plasma and urine LL-37 levels (r = 0.221), even when the data of the cases were analyzed separately. CONCLUSION: LL-37 is notably increased among patients with UTI compared with normal control subjects. Severity of UTI increases the levels of LL-37. The increased level was not only in the patient's urine, but has also been observed in the patient's plasma. Detection of increased levels of LL-37 could help to differentiate subjects with suspected UTI. Accordingly, LL-37 could act as a good marker for diagnosing UTIs.

Elevated levels of the antimicrobial peptide LL-37 in hidradenitis suppurativa are associated with a Th1/Th17 immune response.

Hidradenitis suppurativa (HS) is an inflammatory skin disease with poorly understood immunopathogenic mechanisms. LL-37 is an antimicrobial peptide, which is transcribed from the CAMP (cathelicidin antimicrobial peptide) gene. Previous reports showed upregulated levels of CAMP and LL-37 in HS lesions, and therefore, the aim of this study was to compare levels of LL-37 in HS to other inflammatory skin diseases and to establish immunomodulatory functions of LL-37 in HS. We confirm an upregulation of the LL-37 peptide in lesional HS skin with comparable levels as in psoriasis patients and are able to positively correlate the presence of LL-37 in HS with the presence of T cells, macrophages, neutrophils, IFN-γ, IL-17, IL-23, TNF-α, IL-32 and IL-1ß. Mechanistically, LL-37 boosts the proliferation of unspecifically activated CD4+ T cells via an increased calcium signalling independent of antigen-presenting cells. Targeting LL-37 may therefore represent a new therapeutic option for the treatment of this recalcitrant disease, but it has to be kept in mind that LL-37 also has an antimicrobial function.

The relation of innate and adaptive immunity with viral-induced acute asthma attacks: Focusing on IP-10 and cathelicidin

BACKGROUND: Despite growing evidence suggesting potential association between innate and adaptive immunity in viral-induced acute asthma, there is paucity of data in this area. OBJECTIVE: This study aimed to investigate the association of innate and adaptive immunity with acute asthma attacks by analysing the role of IFN-γ-inducible protein 10 (IP-10), TLR2, cathelicidin, vitamin D and cytokines. MATERIAL AND METHODS: This prospective study included 33 patients with viral-induced acute asthma and 30 children with controlled asthma. Nasopharyngeal swab samples were collected for virus identification and asthma attack scores assessed in acute asthma group. Blood sampling for IP-10, TLR2, cathelicidin, vitamin D levels, and spirometric indices were employed. RESULTS: Serum IP-10 and cathelicidin levels of acute asthma group were significantly higher and vitamin D levels were lower than controlled asthma group (IP-10; p = 0.006, cathelicidin; p = 0.002, vitamin D; p < 0.001). Serum IP-10 levels showed a significant negative correlation with age (p = 0.009), TLR2 (p = 0.05) and spirometric indices (p = 0.002) in all asthmatics and a significant positive correlation with parameters of asthma attack severity (p = 0.03) in acute asthma group. Higher cathelicidin values showed significant positive relation to IP-10 (beta coefficient: 33, p = 0.02). Serum IP-10 levels higher than 38.9pg/ml (sensitivity: 85%, specificity: 47%, p = 0.002) were predictive of virus-induced asthma. Serum IP-10 and vitamin D levels were found to be significantly related to viral-asthma attacks (IP-10; aOR: 8.93, p = 0.03 and vitamin D; aOR: 0.82, p = 0.001). CONCLUSIONS: Innate immunity biomarkers such as serum IP-10 and cathelicidin can be used to predict viral-induced acute asthma. These biomarkers may provide potential new treatment targets for acute asthma

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Antimicrobial peptide levels are linked to airway inflammation, bacterial colonisation and exacerbations in chronic obstructive pulmonary disease.

Antimicrobial peptides (AMPs) are effectors of host defence against infection, inflammation and wound repair. We aimed to study AMP levels in stable chronic obstructive pulmonary disease (COPD) and during acute exacerbations of COPD (AECOPD), and to examine their relation to clinical parameters and inflammatory markers.The 3-year Bergen COPD Cohort Study included 433 COPD patients and 325 controls. Induced sputum was obtained and analysed for levels of the AMPs human cathelicidin (hCAP18/LL-37) and secretory leukocyte protease inhibitor (SLPI), and for the inflammatory markers interleukin (IL)-8, IL-6 and tumour necrosis factor-α (TNF-α) using immunoassays. Systemic hCAP18/LL-37 and vitamin D levels were also studied. Treating AMPs as response variables, non-parametric tests were applied for univariate comparison, and linear regression to obtain adjusted estimates. The risk of AECOPD was assessed by Cox proportional-hazard regression.Sputum AMP levels were higher in patients with stable COPD (n=215) compared to controls (n=45), and further changed during AECOPD (n=56), with increased hCAP18/LL-37 and decreased SLPI levels. Plasma hCAP18/LL-37 levels showed a similar pattern. In stable COPD, high sputum hCAP18/LL-37 levels were associated with increased risk of AECOPD, non-typeable Haemophilus influenzae colonisation, higher age, ex-smoking and higher levels of inflammatory markers.Altered levels of selected AMPs are linked to airway inflammation, infection and AECOPD, suggesting a role for these peptides in airway defence mechanisms in COPD.

Neutrophil extracellular trap formation in the Streptococcus suis-infected cerebrospinal fluid compartment.

Streptococcus suis is an important meningitis-causing pathogen in pigs and humans. Neutrophil extracellular traps (NETs) have been identified as host defense mechanism against different pathogens. Here, NETs were detected in the cerebrospinal fluid (CSF) of S. suis-infected piglets despite the presence of active nucleases. To study NET-formation and NET-degradation after transmigration of S. suis and neutrophils through the choroid plexus epithelial cell barrier, a previously described model of the human blood-CSF barrier was used. NETs and respective entrapment of streptococci were recorded in the "CSF compartment" despite the presence of active nucleases. Comparative analysis of S. suis wildtype and different S. suis nuclease mutants did not reveal significant differences in NET-formation or bacterial survival. Interestingly, transcript expression of the human cathelicidin LL-37, a NET-stabilizing factor, increased after transmigration of neutrophils through the choroid plexus epithelial cell barrier. In good accordance, the porcine cathelicidin PR-39 was significantly increased in CSF of piglets with meningitis. Furthermore, we confirmed that PR-39 is associated with NETs in infected CSF and inhibits neutrophil DNA degradation by bacterial nucleases. In conclusion, neutrophils form NETs after breaching the infected choroid plexus epithelium, and those NETs may be protected by antimicrobial peptides against bacterial nucleases.

Beyond anti-microbial properties: The role of cathelicidin in allergic rhinitis

BACKGROUND: Cathelicidin, an anti-microbial peptide, is a component of the innate immune system. Cathelicidin has anti-microbial, anti-inflammatory and immunoregulatory functions. Knowledge about the role of the innate immune system in the pathogenesis of allergic diseases has expanded in recent years. We measured levels of the LL-37 peptide in the nasal fluids of children with allergic rhinitis (AR) and investigated the possible role of this peptide in the pathogenesis of AR. METHODS: The study population included 46 children who were newly diagnosed with AR and not taking any medication. Thirty-three healthy control subjects were also enrolled. Nasal secretions were collected from the study and control groups using a polyurethane sponge nasal secretion collector, and nasal fluid LL-37 levels were determined using the ELISA method. Results; The levels of LL-37 in the nasal fluid of the AR patients were lower than those of the control group (median of 2.3ng/ml [minimum-maximum, 2.1-3.2] vs. 2.6 ng/ml [2.1-5.4], respectively; p < 0.001), and they were significantly reduced in patients with moderate/severe AR compared with those of patients with mild AR (2.2 ng/ml [2.1-2.4] vs. 2.5 ng/ml [2.1-3.1], respectively; p < 0.001). CONCLUSION: Our results show that children with AR have reduced nasal fluid LL-37 levels compared with healthy controls. Additionally, children with moderate/severe AR have decreased nasal fluid LL-37 levels compared with children with mild AR. These findings highlight the role of cathelicidin in the pathogenesis of AR

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Cathelicidin related antimicrobial peptide, laminin, Toll-like receptors and chemokines levels in experimental hypersensitivity pneumonitis in mice.

INTRODUCTION: Hypersensitivity pneumonitis (HP) is an interstitial lung disease caused by unresolved inflammation and tissue repair pathologies triggered by repeated organic dust exposure. The aim of the study was to investigate changes in levels of the cathelicidin related antimicrobial peptide (CRAMP), laminin (LAM-A1), selected Toll-like receptors (TLR) and chemokines in experimental HP in mice. MATERIALS AND METHODS: Three and 18-month-old female C57BL/6J mice underwent inhalations of the saline extract of Pantoea agglomerans cells, Gram-negative bacterium common in organic dust and known for its pathogenic impact. The inhalations were repeated daily (28 days). ELISA was used for measuring in lung tissue homogenates concentration of CRAMP, LAM-A1, TLR2, TLR4, TLR8, CXCL9 (chemokine [C-X-C motif] ligand) and CXCL10. RESULTS: Levels of TLR2, TLR4 and CXCL9 were significantly higher in both young and old mice lungs already after 7 days of inhalations, while significant increase of LAM-A1 and CXCL10 was noted after 28 days, compared to untreated samples. TLR8 level was significantly augmented only in young mice. Only CRAMP level significantly declined. Significantly higher TLR8 and CXCL9 concentration in untreated samples were noted in old animals compared to young ones. CONCLUSION: Significant alterations of the examined factors levels indicate their role in HP pathogenesis.

On birth single dose live attenuated OPV and BCG vaccination induces gut cathelicidin LL37 responses at 6 week of age: a natural experiment.

In a cross sectional study, we show that infants who received single dose of live attenuated OPV and BCG vaccines within 48h of birth, have higher excretion of human cathelicidin LL37 (p<0.05) in stool at 6wk of age. This response remained unchanged in multivariate analysis after adjusting for sex, mode of delivery, infant age, mother age birth weight and breast milk feeding pattern. This analysis also reveals that irrespective of vaccination, girl infants have higher human-beta-defencin2 (HBD2) and exclusively breastfed infants have higher total and anti-polio specific IgA to all three subtypes in stool (p<0.05). However, vaccination induces anti-polio IgA responses only to infants who are exclusively breastfed. Thus on-birth live attenuated vaccination may provide non-specific beneficial effect against infections while exclusive breastfeeding enhance protection by boosting vaccine induced IgA. The result also suggests that in polio endemic area, exclusive breastfeeding may be sufficient for mucosal anti-polio responses during early infancy.

Cathelicidin and human ß-defensin 2 in bronchoalveolar lavage fluid of children with pulmonary tuberculosis.

BACKGROUND: The antimicrobial peptide cathelicidin LL-37/hCAP-18 and human ß-defensins (hBD) are key factors in innate immune responses of the respiratory tract. OBJECTIVE: To determine LL-37 and hBD-2 concentrations in the bronchoalveolar lavage (BAL) fluid of paediatric patients (aged <16 years) with pulmonary tuberculosis (TB) and to compare these with concentrations in healthy children. METHODS: We measured peptide concentrations using an immunosorbent assay (ELISA). RESULTS Forty TB patients and 40 healthy controls were enrolled in the study (mean age 9.2 ± 4.7 and 8.3 ± 4.2 years, respectively, P = 0.97). The two groups exhibited no statistically significant difference in terms of sex, body mass index, relative weight or 25-hydroxyvitamin D levels. The mean BAL LL-37 level of the TB group was significantly higher than that of the control group (0.95 ± standard deviation [SD] 1.33 vs. 0.35 ± SD 0.51 ng/ml, P = 0.01, t = 2.54). The hBD-2 level was also higher in the TB group; however, the difference was not statistically significant (0.30 ± SD 0.58 vs. 0.14 ± SD 0.30 ng/ml, P = 0.11). There was no correlation between LL-37, hBD-2 and 25-hydroxyvitamin D levels. CONCLUSIONS Our data suggest that LL-37 and hBD-2 may play an important role in TB pathogenesis in children. To our knowledge, this is the first study on BAL LL-37 and hBD-2 concentrations in children with pulmonary TB.

Salivary antimicrobial proteins associate with age-related changes in streptococcal composition in dental plaque.

Secretion of antimicrobial proteins (AMPs) and salivary antibodies can modify biofilm formation at host body surfaces. In adolescents, associations have been reported between dental caries and salivary AMPs. AMPs demonstrate direct antimicrobial effects at high concentrations, and at lower more physiological concentrations they mediate changes in host cell defenses, which may alter the local environment and indirectly shape local biofilm formation. The expression of salivary AMPs in preschool children, at an age when the oral bacteria are known to change, has not been investigated. We sought to investigate salivary AMP expression in the context of previously well-documented changes in the oral cavities of this age group including salivary immunoglobulin A (IgA), oral bacteria and dental caries. Dental plaque and saliva were collected from 57 children aged 12-24 months at baseline, of whom 23 children were followed-up at 3 years of age. At each time, saliva was assessed for LL37, human neutrophil peptides 1-3, calprotectin, lactoferrin, salivary IgA, total plaque bacteria and Streptococcus mutans. Over time, concentrations of AMPs, S. mutans and bacteria-specific salivary IgA increased. Caries experience was also recorded when children were 3 years old. Concentrations of AMPs were highest in the saliva of 3-year-old children with the greatest burden of S. mutans. These data suggest that salivary AMPs are variable over time and between individuals, and are linked with bacterial colonization. At follow up, the majority of children remained caries free. Larger longitudinal studies are required to confirm whether salivary AMP levels are predictive of caries and whether their modulation offers therapeutic benefit.

Expression of cathelicidins mRNA in the goat mammary gland and effect of the intramammary infusion of lipopolysaccharide on milk cathelicidin-2 concentration.

Cathelicidins are a family of antimicrobial peptides found in neutrophils and the epithelium that have broad-spectrum activity against bacteria. This study aimed to investigative the mRNA expression of cathelicidins and protein localization of cathelicidin-2 in the goat mammary gland and its secretion into milk. The mRNA expression of cathelicidins was examined in different regions of the mammary gland by reverse transcription PCR. A cathelicidin-2 antibody was raised in rabbits by immunization with a synthetic cathelicidin-2 peptide consisting of 17 amino acids. The protein localization of cathelicidin-2 was investigated in the mammary gland by immunohistochemistry. Skim milk was collected before (0 h) and 2, 4, 8, 12, and 24h after the intramammary infusion of lipopolysaccharide and saline, and the concentration of cathelicidin-2 was examined by an enzyme immunoassay. The mRNAs of cathelicidin-1, 2, 3, 6, and 7 were expressed in both the teat and deep region of the mammary gland from healthy and mastitic goats. Immunoreactive cathelicidin-2 was not localized in the epithelial cells of the teat skin, teat cistern, or mammary alveoli, whereas it was localized in polymorphonuclear cells in the mammary gland and those collected from the blood and milk. Cathelicidin-2 was detected in skim milk by Western blotting. The concentration of cathelicidin-2 in milk increased 4h after the intramammary infusion of lipopolysaccharide. These results suggest that cathelicidin-2 is expressed in polymorphonuclear cells and is secreted into milk in goat.

LL-37 in periodontal health and disease and its susceptibility to degradation by proteinases present in gingival crevicular fluid.

AIM: To determine the levels of LL-37 in and its susceptibility to degradation by components of gingival crevicular fluid (GCF) in periodontal health and disease. MATERIALS AND METHODS: Levels of LL-37 in GCF from periodontitis patients and periodontally healthy subjects were determined by ELISA. In addition, degradation of synthetic/exogenous LL-37 by components of GCF in the presence and absence of inhibitors was determined by matrix-assisted laser desorption/ionization time of flight mass spectrometry. RESULTS: The concentration of native LL-37 in GCF from Porphyromonas gingivalis positive (Pg+) and P. gingivalis negative (Pg-) sites in periodontitis patients was significantly higher than in GCF from healthy subjects. When synthetic LL-37 was added to healthy GCF, the peptide was not degraded. Conversely, GCF from Pg+ sites rapidly degraded synthetic LL-37 which was prevented in the presence of Arg- and Lys- gingipain inhibitors. Synthetic LL-37 was degraded more slowly by GCF from Pg- sites. CONCLUSIONS: LL-37 is detectable in GCF in periodontal health and disease. The rapid degradation of synthetic LL-37 in periodontitis GCF, particularly in Pg+ sites, limits its role as a potential therapeutic in the gingival crevice. These results highlight the need to design stable peptide mimetics of LL-37 as future therapeutics in periodontitis.

Human colonic mucus is a reservoir for antimicrobial peptides.

BACKGROUND AND AIMS: To prevent bacterial adherence and translocation, the colonic mucosa is covered by a protecting mucus layer and the epithelium synthesizes antimicrobial peptides. The present qualitative study investigated the contents and interaction of these peptides in and with rectal mucus. METHODS: Rectal mucus extracts were analyzed for antimicrobial activity and screened with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Dot blot and immunohistochemistry for antimicrobial peptides. In addition, binding of AMPs to mucins was investigated by Western blot and enzyme-linked lectin assays. RESULTS: In functional tests the mucus layer exhibited a strong antimicrobial activity. We detected 11 antimicrobial peptides in mucus extracts from healthy persons including the defensins HBD-1 and -3, the cathelicidin LL-37, ubiquitin, lysozyme, histones, high mobility group nucleosome-binding domain-containing protein 2, ubiquicidin and other ribosomal proteins. AMPs were bound by mucins but this was demonstrated to be reversible and inhibition of antibacterial activity was limited. CONCLUSION: These findings indicate that epithelial antimicrobial peptides are retained in the intestinal mucus layer without losing their efficacy. Thus, the mucus layer and its composition provide an attractive drug target to restore antimicrobial barrier function in intestinal diseases.

Salivary concentration of the antimicrobial peptide LL-37 in children.

OBJECTIVE: Antimicrobial peptides are important components of innate immunity, especially in the unique environment of the oral cavity. Lack of the human cathelicidin LL-37 has been implicated in severe periodontitis, whilst high salivary levels of LL-37 seem to increase caries resistance. Limited data exists about the concentration of LL-37 in saliva of young children. In this study, the salivary concentration of LL-37 was examined in relation to age, gender, type of dentition (primary, mixed or permanent) and caries experience of children. DESIGN: Unstimulated whole saliva was collected from 49 systemically healthy and gingivitis free children aged 2-18 years old. Their caries activity was recorded. The salivary LL-37 concentration was determined by enzyme linked immunosorbent assay (ELISA). RESULTS: LL-37 was detected in all saliva samples. Its concentration varied widely, with girls exhibiting higher peptide levels than boys. A positive correlation of the LL-37 concentration was observed with age. Children with primary dentition had significantly lower peptide concentration than those with mixed or permanent dentition. Significantly lower concentrations of LL-37 were also found in children with high caries activity, compared to caries free children or to children with low to moderate caries activity. CONCLUSIONS: Our results reinforce the belief that LL-37 is an important molecule of immunity in the oral environment and it seems to play a protective role against caries.

Evaluation of protein expression in bovine bronchoalveolar fluid following challenge with Mannheimia haemolytica.

Proteomics analysis of bovine bronchoalveolar fluid (BAF) following induction of pneumonia with Mannheimia haemolytica using nanoflow liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) resulted in the identification of 88 unique proteins. Proteins detected in BAF included antimicrobial peptides (AMPs), complement factors, acute-phase proteins, protease inhibitors, and proteins involved in oxidation-reduction. Notwithstanding biological variation, differences in relative protein abundance, determined using normalized peptide counts, were detected for select proteins in BAF from genuinely infected versus sham-infected animals. To demonstrate the applicability of using normalized peptide counts to assess protein expression trends, LC-MS/MS data for the acute-phase protein haptoglobin (HPT) were compared with ELISA data, and statistical evaluation of the relationship between the data revealed a strong measure of association. Differences were detected between sham- and genuinely infected animals for haptoglobin, as well as the AMPs cathelicidin-1 and cathelicidin-4, and inter-α-trypsin inhibitor heavy chain-4, a fairly novel protein involved in the acute phase response. Though the small sample size limited the scope of the inferences, the results indicate the likely importance of AMPs and acute-phase proteins during respiratory infection, and provide additional information regarding potential mechanisms involved in the bovine mucosal barrier defense.