RESUMEN

Periodontal disease is triggered by surface bacterial biofilms where bacteria are less susceptible to antibiotic treatment. The development of liposome-based delivery mechanisms for the therapeutic use of antimicrobial peptides is an attractive alternative in this regard. The cationic antimicrobial peptide LL-37 (human cathelicidin) is well-known to exert antibacterial activity against P orphyromonas gingivalis, a keystone oral pathogen. However, the antibacterial activity of the 16-amino acid fragment (LL17-32) of LL-37, is unknown. In addition, there are still gaps in studies using liposomal formulations as delivery vehicles of antibacterial peptides against this pathogen. This study was designed to examine the influence of the different types of liposomal formulations to associate and deliver LL17-32 to act against P. gingivalis. Chitosans of varying Mw and degree of acetylation (DA) were adsorbed at the surface of soya lecithin (SL) liposomes. Their bulk (average hydrodynamic size, ζ-potential and membrane fluidity) and ultrastructural (d-spacing, half-bilayer thickness and the water layer thickness) biophysical properties were investigated by a panel of techniques (DLS, SAXS, M3-PALS, fluorescence spectroscopy and TEM imaging). Their association efficiency, in vitro release, stability, and efficacy in killing the periodontal pathogen P. gingivalis were also investigated. All liposomal systems possessed spherical morphologies and good shelf-life stabilities. Under physiological conditions, chitosan formulations with a high DA demonstrated enhanced stability in comparison to low DA-chitosan formulations. Chitosans and LL17-32 both decreased SL-liposomal membrane fluidity. LL17-32 exhibited a high degree of association with SL-liposomes without in vitro release. In biological studies, free LL17-32 or chitosans alone, demonstrated microbicidal activity against P. gingivalis, however this was attenuated when LL17-32 was loaded onto the SL-liposome delivery system, presumably due to the restrained release of the peptide. A property that could be harnessed in future studies (e.g., oral mucoadhesive slow-release formulations).

RESUMEN

The only human cathelicidin, LL-37, is a host defense antimicrobial peptide with antimicrobial activities against protozoans, fungi, Gram(+) and Gram(-) bacteria, and enveloped viruses. It has been shown in experiments in vitro that LL-37 is able to induce the production of various inflammatory and anti-inflammatory cytokines and chemokines by different human cell types. However, it remains an open question whether such cytokine induction is physiologically relevant, as LL-37 exhibited its immunomodulatory properties at concentrations that are much higher (>20 µg/mL) than those observed in non-inflamed tissues (1-5 µg/mL). In the current study, we assessed the permeability of LL-37 across the Caco-2 polarized monolayer and showed that this peptide could pass through the Caco-2 monolayer with low efficiency, which predetermined its low absorption in the gut. We showed that LL-37 at low physiological concentrations (<5 µg/mL) was not able to directly activate monocytes. However, in the presence of polarized epithelial monolayers, LL-37 is able to activate monocytes through the MAPK/ERK signaling pathway and induce the production of cytokines, as assessed by a multiplex assay at the protein level. We have demonstrated that LL-37 is able to fulfill its immunomodulatory action in vivo in non-inflamed tissues at low physiological concentrations. In the present work, we revealed a key role of epithelial-immune cell crosstalk in the implementation of immunomodulatory functions of the human cathelicidin LL-37, which might shed light on its physiological action in vivo.

Asunto(s)

RESUMEN

The parasite Trichomonas vaginalis is the aetiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease worldwide. This infection often remains asymptomatic and is related to several health complications. The traditional treatment for trichomoniasis uses drugs of the 5-nitroimidazole family, such as metronidazole; however, scientific reports indicate an increasing number of drug-resistant strains. Antimicrobial peptides could be an alternative or complementary treatment. In this sense, one attractive candidate is the human cathelicidin, being LL-37 its active form. LL-37 possesses microbicidal activity against many microorganisms such as bacteria, Candida albicans, and Entamoeba histolytica. Shorter sequences derived from this peptide, such as KR-20, FK-13 and KR-12, have been shown to possess a higher microbicidal effect than LL-37. In this study, we determined the activity of LL-37 and its derivatives against T. vaginalis, which was unknown. The results showed that the four peptides (LL-37, KR-20, FK-13-NH2 and KR-12) decreased the viability of T. vaginalis on a 5-nitroimidazole-sensitive and a 5-nitroimidazole-resistant strain; however, KR-20 was the most effective peptide, followed by FK-13-NH2. Low concentrations of all peptides showed a better effect when combined with metronidazole in the sensitive and resistant T. vaginalis strains. These results are promising for potential future therapeutic uses.

Asunto(s)

RESUMEN

Hyaline cartilage is an important tracheal structure, yet little is known about its molecular composition, complicating investigation of pathologies and replacement options. Our aim was to research tracheal hyaline cartilage structure, protective tissue factors and variations in healthy humans. The tissue material was obtained from 10 cadavers obtained from the Riga Stradins University Institute of Anatomy and Anthropology archive. Tissues were stained with Bismarck brown and PAS for glycosaminoglycans, and immunohistochemistry was performed for HBD-2, HBD-3, HBD-4, IL-10 and LL-37. The slides were inspected by light microscopy and Spearman's rank correlation coefficient was calculated. The extracellular matrix was positive across hyaline cartilage for PAS, yet Bismarck brown marked positive proliferation and growth zones. Numerous positive cells for both factors were found in all zones. All of the antimicrobial defence molecules and cytokines were found in a moderate number of cells, except in the mature cell zone with few positive cells. Spearman's rank correlation coefficient revealed strong and moderate correlations between studied factors. Hyaline cartilage is a tracheal defence structure with a moderate number of antimicrobial defence protein and cytokine immunoreactive cells as well as numerous glycosaminoglycan positive cells. The extracellular matrix glycosaminoglycans provide structural scaffolding and intercellular signalling. The correlations between the studied factors confirm the synergistic activity of them.

RESUMEN

The mechanisms for maintaining oral cavity homeostasis are subject to the constant influence of many environmental factors, including various chemicals and microorganisms. Most of them act directly on the oral mucosa, which is the mechanical and immune barrier of the oral cavity, and such interaction might lead to the development of various oral pathologies and systemic diseases. Two important players in maintaining oral health or developing oral pathology are the oral microbiota and various immune molecules that are involved in controlling its quantitative and qualitative composition. The LL-37 peptide is an important molecule that upon release from human cathelicidin (hCAP-18) can directly perform antimicrobial action after insertion into surface structures of microorganisms and immunomodulatory function as an agonist of different cell membrane receptors. Oral LL-37 expression is an important factor in oral homeostasis that maintains the physiological microbiota but is also involved in the development of oral dysbiosis, infectious diseases (including viral, bacterial, and fungal infections), autoimmune diseases, and oral carcinomas. This peptide has also been proposed as a marker of inflammation severity and treatment outcome.

RESUMEN

This study aims to push the frontier of the engineering of human cathelicidin LL-37, a critical antimicrobial innate immune peptide that wards off invading pathogens. By sequential truncation of the smallest antibacterial peptide (KR12) of LL-37 and conjugation with fatty acids, with varying chain lengths, a library of lipopeptides is generated. These peptides are subjected to antibacterial activity and hemolytic assays. Candidates (including both forms made of l- and d-amino acids) with the optimal cell selectivity are subsequently fed to the second layer of in vitro filters, including salts, pH, serum, and media. These practices lead to the identification of a miniature LL-37 like peptide (d-form) with selectivity, stability, and robust antimicrobial activity in vitro against both Gram-positive and negative bacteria. Proteomic studies reveal far fewer serum proteins that bind to the d-form than the l-form peptide. C10-KR8d targets bacterial membranes to become helical, making it difficult for bacteria to develop resistance in a multiple passage experiment. In vivo, C10-KR8d is able to reduce bacterial burden of methicillin-resistant Staphylococcus aureus (MRSA) USA300 LAC in neutropenic mice. In addition, this designer peptide prevents bacterial biofilm formation in a catheter-associated mouse model. Meanwhile, C10-KR8d also recruits cytokines to the vicinity of catheters to clear infection. Thus, based on the antimicrobial region of LL-37, this study succeeds in identifying the smallest anti-infective peptide C10-KR8d with both robust antimicrobial, antibiofilm, and immune modulation activities.

Asunto(s)

RESUMEN

Nanotechnology-based therapeutic approaches have attracted attention of scientists, in particular due to the special features of nanomaterials, such as adequate biocompatibility, ability to improve therapeutic efficiency of incorporated drugs and to limit their adverse effects. Among a variety of reported nanomaterials for biomedical applications, metal and metal oxide-based nanoparticles offer unique physicochemical properties allowing their use in combination with conventional antimicrobials and as magnetic field-controlled drug delivery nanocarriers. An ever-growing number of studies demonstrate that by combining magnetic nanoparticles with membrane-active, natural human cathelicidin-derived LL-37 peptide, and its synthetic mimics such as ceragenins, innovative nanoagents might be developed. Between others, they demonstrate high clinical potential as antimicrobial, anti-cancer, immunomodulatory and regenerative agents. Due to continuous research, knowledge on pleiotropic character of natural antibacterial peptides and their mimics is growing, and it is justifying to stay that the therapeutic potential of nanosystems containing membrane active compounds has not been exhausted yet.

Asunto(s)

RESUMEN

Staphylococcus epidermidis is a common nosocomial pathogen able to form biofilms in indwelling devices, resulting in chronic infections, which are refractory to antibiotics treatment. Staphylococcal biofilms are also associated with the delayed reepithelization and healing of chronic wounds. The human cathelicidin peptide LL-37 has been proven active against S. epidermidis biofilms in vitro and to promote wound healing. As previous studies have demonstrated that fragments of LL-37 could possess an equal antibacterial activity as the parent peptide, we tested whether shorter (12-mer) synthetic fragments of LL-37 maintained the antibiofilm and/or immune modulating activity, aiming at the identification of essential regions within the LL-37 parent sequence. Three fragments of LL-37 displayed improved activity against S. epidermidis in terms of biofilm inhibition and eradication, a reduced cytotoxicity to human keratinocytes and erythrocytes. In addition, KR-12 and VQ-12V26 enhanced wound healing potential, relative to LL37. FK-12 and KR-12 are truncated version of the cathelicidin, previously reported as valid antimicrobials, whereas VQ-12V26 is a single substituted LL-37 fragment. Remarkably, the single substitution aspartic acid to valine in position 26 caused gain of antimicrobial function in the inactive VQ-12 fragment. The combination of antibiofilm, wound healing potential, and low cytotoxicity makes KR-12 and VQ-12V26 promising therapeutic agents and lead compounds for further improvement and understanding of antibiofilm and wound healing properties.

Asunto(s)

RESUMEN

Astragalus polysaccharides (APS), one of the major active components in Astragalus membranaceus, is an effective immunomodulator used in the treatment of immunological diseases in China. However, the anti-infective action and mechanism of APS is not fully known. In the present study, we found that APS induced the expression of human cathelicidin antimicrobial peptide LL-37, a key host anti-infective molecule, in both mRNA and protein levels in respiratory epithelial cells HBE16 and A549. Furthermore, the lysate and supernatant from APS-treated HBE16 cells both exhibited an obvious antibacterial action, which was partially neutralizated by LL-37 monoclonal antibody. In addition, APS also significantly elevated the phosphorylation of p38 MAPK and JNK and caused the degradation of IκBα. Specific inhibitors of p38 MAPK, JNK, or NF-κB obviously abolished APS-induced LL-37 synthesis and antibacterial activity, respectively. Taken together, our results confirmed the enhancement of APS on LL-37 induction and antibacterial action in respiratory epithelial cells, which may be attributed to activation of p38 MAPK/JNK and NF-κB pathways. Furthermore, these results also supported the clinical application of APS in the treatment of infectious diseases.

Asunto(s)

RESUMEN

Dyslipidemia in patients with hypercholesterolemia has been recently linked to increased human cathelicidin LL-37 (LL-37) serum concentration. We tested a hypothesis that upregulated expression of LL-37 gene in peripheral blood leucocytes is involved in dyslipidemia in patients with hypercholesteremia. Patients with hypercholesterolemia were used in the study. Expression of LL-37 and human glyceraldehyde-3-phosphate dehydrogenase in peripheral blood leucocytes were quantified by real-time RT-PCR. Serum LL-37 concentration was estimated by enzyme-linked immunosorbent assay. Serum lipid levels were assessed by absorptiometry in all cases. Patients with hypercholesterolemia as compared to control ones were characterized by (a) an up-regulation of LL-37 gene expression in peripheral blood leucocytes with parallel increase of serum LL-37 concentration and (b) an increase of serum total and low-density lipoprotein cholesterol concentrations. Patients with hypercholesterolemia after a treatment with atorvastatin calcium 20 mg daily as compared to that patients before the treatment: an down-regulation of LL-37 gene expression in peripheral blood leucocytes with parallel decrease of serum LL-37 concentration. We also found significant correlation between serum LL-37 and high-density lipoprotein cholesterol levels (r = 0.7290, P < 0.0001). The results suggest that hypercholesterolemia is associated with an increased LL-37 gene expression in peripheral blood leucocytes. The correlation between serum LL-37 and high-density lipoprotein cholesterol levels suggests that LL-37 may play a key role in regulation of cholesterol levels in hypercholesterolemia.

Asunto(s)

RESUMEN

BACKGROUND: Active Vitamin D analogues are used clinically for prevention and treatment of secondary hyperparathyroidism (SHPT) in hemodialysis (HD) patients. Nutritional vitamin D supplementation is used for additional local parathyroid (PTH) suppression, with lower incidence of hypercalcemia and hyperphosphatemia. This study evaluates the possible beneficial effects of combined vitamin D treatment (paricalcitol and cholecalciferol). METHODS: Sixty HD patients with serum parathyroid hormone (iPTH) >300 pg/mL were enrolled. All patients administered 2 mcg/day of paricalcitol and were randomly allocated into control group (placebo) or study group (cholecalciferol) for 16 weeks. Serum 25(OH)D3, iPTH and human cathelicidin (hCAP-18) were measured at baseline and during follow-up. RESULTS: iPTH levels decreased in the study group appropriately and were more significantly decreased at 16 weeks. Study group had significantly increased 25(OH)D3 levels. In addition, the study group had significantly increased serum hCAP-18 levels compared with control group. Correlation analysis showed a significant correlation between the percentage increase in serum hCAP-18 and 25(OH)D3 levels. CONCLUSIONS: Cholecalciferol, in combination with paricalcitol, additively lowers the iPTH levels in a significant number of patients after 16 weeks of supplementation. A dose of 5000 IU/week of cholecalciferol could maintain serum 25(OH)D3 levels above 30 ng/dL as early as 8 weeks after beginning supplementation. Doubling of serum cathelicidin levels were noted after 16 weeks of cholecalciferol supplementation in 40% of study patients.

Asunto(s)

RESUMEN

OBJECTIVE: Controversies exist regarding the relationship between the concentrations of antimicrobial peptides (AMPs) and presence of dental caries in children. Thus, the aim of this study was to examine levels of AMPs in saliva of caries-free (CF), early childhood caries (ECC) and severe early childhood caries (S-ECC) children to determine if the levels of these salivary peptides individually or in combinations were related to caries severity and mutans streptococci levels. DESIGN: 36 to 60 month-old children were selected to participate in this study. Children were grouped into CF group (n=29), ECC group (n=25) and S-ECC group (n=29). Saliva was collected from children for microbiological analysis by culture. Salivary concentrations of cathelicidin LL-37, human ß-defensin 2 (hBD-2), human ß-defensin 3 (hBD-3) and histatin-5 (HTN-5) were determined by ELISA. RESULTS: Salivary concentrations of AMPs did not differ among CF, ECC and S-ECC groups. Data showed positive correlations between mutans streptococci levels and salivary hBD-2 or HTN-5. Positive correlations were found between hBD-2, hBD-3, LL-37 and HTN-5. Combinations among AMPs, mainly LL-37, were positively associated with caries levels. CONCLUSIONS: Salivary concentrations of AMPs individually were not associated with the severity of early childhood caries. The stimulus of caries appears to trigger a biological response, however, with a combination of these peptides.

Asunto(s)

RESUMEN

The ribonuclease activity of the soluble glycoprotein E(rns) of pestiviruses represents a unique mechanism to circumvent the host's innate immune system by blocking interferon type-I synthesis in response to extracellularly added single- (ss) and double-stranded (ds) RNA. However, the reason why pestiviruses encode a ribonuclease in addition to the abundant serum RNases remained elusive. Here, we show that the 5' UTR and NS5B regions of various strains of the RNA genome of the pestivirus bovine viral diarrhea virus (BVDV) are resistant to serum RNases and are potent TLR-3 agonists. Inhibitory activity of E(rns) was restricted to cleavable RNA products, and did not extend to the synthetic TLR-7/8 agonist R-848. RNA complexed with the antimicrobial peptide LL37 was protected from degradation by E(rns)in vitro but was fully inhibited by E(rns) in its ability to induce IFN in cell cultures, suggesting that the viral protein is mainly active in cleaving RNA in an intracellular compartment. We propose that secreted E(rns) represents a potent IFN antagonist, which degrades viral RNA that is resistant to the ubiquitous host RNases in the extracellular space. Thus, the viral RNase prevents its own pathogen-associated molecular pattern (PAMP) to inadvertently activate the IFN response that might break innate immunotolerance required for persistent pestivirus infections.

Asunto(s)

RESUMEN

We have synthesized 39 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] analogs having two side chains attached to carbon-20 (Gemini) with various modifications and compared their anticancer activities. Five structure-function rules emerged to identify analogs with enhanced anticancer activity. One of these active analogs, BXL-01-0126, was more potent than 1,25(OH)2D3 in mediating 50% clonal inhibition of cancer cell growth. Murine studies found that BXL-01-0126 and 1,25(OH)2D3 had nearly the same potency to raise serum calcium levels. Taken together, BXL-01-0126 when compared to 1,25(OH)2D3 has greater anticancer potency, but similar toxicity causing hypercalcemia. We focused on the effect of these compounds on the stimulation of expression of human cathelicidin antimicrobial peptide (CAMP) whose gene has a vitamin D response element in its promoter. Expression of CAMP mRNA and protein increased in a dose-response fashion after exposure of acute myeloid leukemia (AML) cells to the Gemini analog, BXL-01-126, in vitro. A xenograft model of AML was developed using U937 AML cells injected into NSG-immunodeficient mice. Administration of vitamin D3 compounds to these mice resulted in substantial levels of CAMP in the systemic circulation. This suggests a unique prophylactic treatment at diagnosis or during induction chemotherapy for AML patients to provide them with protection against various microbial infections through CAMP induction.

Asunto(s)

RESUMEN

KR-12 (residues 18-29 of LL-37) was known to be the smallest peptide of human cathelicidin LL-37 possessing antimicrobial activity. In order to optimize α-helical short antimicrobial peptides having both antimicrobial and antiendotoxic activities without mammalian cell toxicity, we designed and synthesized a series of KR-12 analogs. Highest hydrophobic analogs KR-12-a5 and KR-12-a6 displayed greater inhibition of lipopolysaccharide (LPS)-stimulated tumor necrosis factor-α production and higher LPS-binding activity. We have observed that antimicrobial activity is independent of charge, but LPS neutralization requires a balance of hydrophobicity and net positive charge. Among KR-12 analogs, KR-12-a2, KR-12-a3 and KR-12-a4 showed much higher cell specificity for bacteria over erythrocytes and retained antiendotoxic activity, relative to parental LL-37. KR-12-a5 displayed the strongest antiendotoxic activity but almost similar cell specificity as compared with LL-37. Also, these KR-12 analogs (KR-12-a2, KR-12-a3, KR-12-a4 and KR-12-a5) exhibited potent antimicrobial activity (minimal inhibitory concentration: 4 µM) against methicillin-resistant Staphylococcus aureus. Taken together, these KR-12 analogs have the potential for future development as a novel class of antimicrobial and anti-inflammatory therapeutic agents.

Asunto(s)

RESUMEN

Objective To reconstruct the proteinic sequence of human cathelicidin LL-37 to increase the bactericidal activity of LL-37 and to express the reconstructed LL-37 (rLL-37) in bacterium. Methods The two dimensional structure, three dimensional structure and chemical characteristic of LL-37 were analyzed by Soft Ware Anthepro 5.0 and SWISS-MODEL. Without the three dimensional changes of LL-37, some negative amino acids of human cathelicidin LL-37 were replaced by positive amino acids and the positive charge of LL-37 was increased. According to the proteinic sequence changes of rLL-37, the DNA sequence of rLL-37 was reconstructed by Touch-Down PCR and recombined with vector pET-28a (+), thus rLL-37 was expressed in E.coli. BL21 (DE3) by the induction of IPTG and was purified by chromatography. Results Glu~ 16 , Asp~ 26 , Glu~ 36 of LL-37 were replaced by Gln~ 16 , Asn~ 26 , Gln~ 36 and the static charge of LL-37 was increased from +5.8 to +9.0 at pH 7.4. The DNA sequence of rLL-37 was reconstructed and inserted into vector pET-28a (+), the rLL-37 was expressed in E.coli. BL21 (DE3) and purified by strong cation exchange supports Macro-Prep High S successfully. The rLL-37 was proved by the means of inhibitory zone to be able to kill Gram-negative bacteria and Gram-positive bacteria. Conclusion It is feasible to reconstruct human cathelicidin LL-37 and express the protein in bacteria by fusion, which make it possible to produce more rLL-37 and study its biological function deeply.

RESUMEN

Objective To employ 2 approaches to construct and express reconstructed LL-37 (rLL-37) in procaryotic system, and to explore a better preparation method. Methods The first method: the rLL-37 was inserted into vector pET-28a (+), then was induced to express in E.coli. BL21 (DE3) and purified by chromatography; the second method: the rare codons in the rLL-37 gene sequence were substituted by the preferred codons of procaryotic cell, and a fragment of carrier protein molecule (CPM) was added to the N termination of the objective sequence to construct expression plasmid pET-30a(+)-CPM-rLL-37, then the rLL-37 was expressed in E.coli. BL21 Star(DE3) and purified by chromatography. The productive rates of the 2 methods were compared and the antimicrobial effects of obtained rLL-37 was studied. Results The first method: the DNA sequence of rLL-37 was obtained successively by Touch-Down PCR. The expression plasmid pET-30a(+)-CPM-rLL-37 was expressed with fusion protein in E.coli BL21 (DE3). The expression rate accounted for 20% of total bacterio-protein, then the expressed product was purified by using high positive ion exchange column Macro-Prep High S; The second method: a fragment of carrier protein molecule was designed that contained 28 amino-acid residue and its pHi was 2.7, net charge was-6.0 at pH 7.4. After the expression plasmid pET-30a(+)-CPM-rLL-37 was constructed successively, it was expressed in E.coli BL21 Star (DE3). The expressed fusion protein accounted for 35% of total bacterio-protein, then the expressed product was purified by using affinity binding chromatography with TALON resins successfully. The obtained 2 kinds of rLL-37 were able to kill both Gram-negative and-positive bacteria by the means of inhibitory zone. Conclusion It’s feasible to prepare efficiently rLL-37 in procaryotic system, which founds the basis for the further research on bactericidal activity of rLL-37.