Objective: To fabricate TiO₂ nanotube material functionalized by antimicrobial peptide LL-37, and to explore its effects on biological behaviors such as adhesion and migration of human keratinocytes (HaCaT) and its antibacterial properties. Methods: The TiO₂ nanotube array (NT) was constructed on the surface of polished titanium (PT) by anodization, and the antimicrobial peptide LL-37 was loaded on the surface of TiO₂ nanotube (LL-37/NT) by physical adsorption. Three samples were selected by simple random sampling in each group. Surface morphology, roughness, hydrophilicity and release characteristics of LL-37 of the samples were analyzed with a field emission scanning electron microscope, an atomic force microscope, a contact angle measuring device and a microplate absorbance reader. HaCaT cells were respectively cultured on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of cell was observed by field emission scanning electron microscope. The number of cell adhesion was observed by cellular immunofluorescence staining. Cell counting kit-8 (CCK-8) assay was used to detect cell proliferation. Wound scratch assay was used to observe the migration of HaCaT. The above experiments were used to evaluate the effect of each group on the biological behavior of HaCaT cells. To evaluate their antibacterial effects, Porphyromonas gingivalis (Pg) was respectively inoculated on the surface of three groups of titanium samples. Each group had 3 replicates. The morphology of bacteria was observed by field emission scanning electron microscope. Bacterial viability was determined by live/dead bacterial staining. Results: A uniform array of nanotubes could be seen on the surface of titanium samples in LL-37/NT group, and the top of the tube was covered with granular LL-37. Compared with PT group [the roughness was (2.30±0.18) nm, the contact angle was 71.8°±1.7°], the roughness [(20.40±3.10) and (19.10±4.11) nm] and hydrophilicity (the contact angles were 22.4°±3.1° and 25.3°±2.2°, respectively) of titanium samples increased in NT and LL-37/NT group (P<0.001). The results of in vitro release test showed that the release of antimicrobial peptide LL-37 was characterized by early sudden release (1-4 h) and long-term (1-7 d) slow release. With the immunofluorescence, more cell attachment was found on NT and LL-37/NT than that on PT at the first 0.5 and 2.0 h of culture (P<0.05). The results of CCK-8 showed that there was no significant difference in the proliferation of cells among groups at 1, 3 and 5 days after culture. Wound scratch assay showed that compared with PT and NT group, the cell moved fastest on the surface of titanium samples in LL-37/NT group at 24 h of culture [(96.4±4.9)%] (F=35.55, P<0.001). A monolayer cells could be formed and filled with the scratch in 24 h at LL-37/NT group. The results of bacterial test in vitro showed that compared with the PT group, the bacterial morphology in the NT and LL-37/NT groups was significantly wrinkled, and obvious bacterial rupture could be seen on the surface of titanium samples in LL-37/NT group. The results of bacteria staining showed that the green fluorescence intensity of titanium samples in LL-37/NT group was the lowest in all groups (F=66.54,P<0.001). Conclusions: LL-37/NT is beneficial to the adhesion and migration of HaCaT cells and has excellent antibacterial properties, this provides a new strategy for the optimal design of implant neck materials.
Humans ; Titanium/chemistry* ; Antimicrobial Peptides ; Cathelicidins ; Sincalide ; Anti-Bacterial Agents/pharmacology* ; Nanotubes/chemistry* ; Dental Materials ; Bacteria ; Keratinocytes ; Surface Properties

Campylobacter is a worldwide foodborne pathogen, associated with human gastroenteritis. The efficient translocation of Campylobacter and its ability to secrete toxins into host cells are the 2 key features of Campylobacter pathophysiology which trigger inflammation in intestinal cells and contribute to the development of gastrointestinal symptoms, particularly diarrhoea, in humans. The purpose of conducting this literature review is to summarise the current understanding of: i) the human immune responses involved in the elimination of Campylobacter infection and ii) the resistance potential in Campylobacter against these immune responses. This review has highlighted that the intestinal epithelial cells are the preliminary cells which sense Campylobacter cells by means of their cell-surface and cytosolic receptors, activate various receptors-dependent signalling pathways, and recruit the innate immune cells to the site of inflammation. The innate immune system, adaptive immune system, and networking between these systems play a crucial role in bacterial clearance. Different cellular constituents of Campylobacter, mainly cell membrane lipooligosaccharides, capsule, and toxins, provide protection to Campylobacter against the human immune system mediated killing. This review has also identified gaps in knowledge, which are related to the activation of following during Campylobacter infection: i) cathelicidins, bactericidal permeability-increasing proteins, chemokines, and inflammasomes in intestinal epithelial cells; ii) siglec-7 receptors in dendritic cell; iii) acute phase proteins in serum; and iv) T-cell subsets in lymphoid nodules. This review evaluates the existing literature to improve the understanding of human immunity against Campylobacter infection and identify some of the knowledge gaps for future research.
Acute-Phase Proteins ; Antigen-Presenting Cells ; Campylobacter Infections ; Campylobacter ; Cathelicidins ; Cell Membrane ; Chemokines ; Cytosol ; Dendritic Cells ; Epithelial Cells ; Gastroenteritis ; Guillain-Barre Syndrome ; Homicide ; Humans ; Immune System ; Inflammasomes ; Inflammation ; T-Lymphocyte Subsets ; Toll-Like Receptors

OBJECTIVE: To discuss the clinical significance of antibacterial peptide LL-37 in the early diagnosis of patients with sepsis in emergency department. METHODS: Forty patients diagnosed with sepsis in the emergency department of the Affiliated Hospital of Zunyi Medical College from December 2017 to March 2018 were enrolled as sepsis group. Twenty healthy volunteers were enrolled contemporaneously in our hospital at medical center as healthy control group. Peripheral blood was collected immediately after diagnosis in sepsis group or during physical examination in healthy control group. The expression of antibacterial peptide LL-37 was determined by enzyme-linked immunosorbent assay (ELISA). Meanwhile, serum procalcitonin (PCT) and C-reactive protein (CRP) levels were determined. The differences in antibacterial peptide LL-37, PCT and CRP levels between the two groups were compared. Pearson correlation method was used to analyze the correlation between antibacterial peptide LL-37, PCT and CRP. Receiver operating characteristic (ROC) curve was drawn, and the early individually or jointly diagnostic value of each detected index for sepsis was analyzed. RESULTS: The levels of antimicrobial peptide LL-37, PCT and CRP in peripheral blood of sepsis group were significantly higher than those of healthy control group [LL-37 (μg/L): 1.34±0.69 vs. 0.10±0.06, PCT (μg/L): 46.67±39.51 vs. 0.03±0.02, CRP (mg/L): 129.68±49.83 vs. 3.16±2.85], with statistically significant differences (all P < 0.05). Pearson correlation analysis showed that the expression of antimicrobial peptide LL-37 was positively correlated with PCT and CRP levels (r₁ = 0.835, r₂ = 0.932, both P < 0.01). ROC curve analysis showed that the area under ROC curve (AUC) of LL-37, PCT and CRP for early diagnosis of sepsis was 0.885, 0.963 and 0.983, respectively, and the AUC of combined diagnosis of the three parameters was as high as 0.994, indicating that the value of combined diagnosis of sepsis was greater than that of single diagnosis; when the combined prediction probability of the three parameters was 0.92, the sensitivity was 97.5%, and the specificity was 95.0%. CONCLUSIONS Antibacterial peptide LL-37 has certain clinical value in early diagnosis of patients with sepsis, which can be used as early routine monitoring indicators for patients with early sepsis when combined with PCT and CRP.
Antimicrobial Cationic Peptides/metabolism* ; C-Reactive Protein ; Calcitonin ; Calcitonin Gene-Related Peptide ; Early Diagnosis ; Emergency Service, Hospital ; Humans ; Protein Precursors ; ROC Curve ; Sepsis/metabolism* ; Cathelicidins

Infection is one of the main causes of death in clinical patients, and multi-drug resistance leads to ineffective treatment with conventional antibiotics. Therefore, it is imperative to develop new anti-infective drugs. Antimicrobial peptides cathelicidins are cationic host defense peptides found in many organisms. It has been demonstrated by in vivo and in vitro studies that antimicrobial peptides cathelicidins not only show broad-spectrum antibacterial activity and high sensitivity to drug-resistant bacteria, but also have a good guiding effect on the immune response. This paper summarizes the reports of antimicrobial peptides cathelicidins in recent years, highlighting their research achievements in antibiosis, anti-inflammatory, chemotaxis regulation and phagocytosis, providing new ideas for the treatment of infection-related diseases.
Anti-Bacterial Agents ; Anti-Infective Agents ; Antimicrobial Cationic Peptides ; Bacteria ; Cathelicidins ; Humans

The platelet-rich plasma (PRP) and antimicrobial peptides neutrophil extract (AMP extract) were prepared from rabbit neutrophils as two autologous blood-derived preparations, which could be applied locally to enhance healing process of tissues. Both preparations were analyzed using the MALDI TOF method for accurate qualitative assay. Growth factors (PDGF and VEGF) and microbicidal protein were reported in PRP. In AMP extract a-defensins, namely; NP-1, -2, -3a, -3b, -4, and -5 and cathelicidins represented among other by 15-kDa antibacterial protein (p15s) were detected. In the second part of our study the influence of antimicrobial extract on macrophages in vitro was tested. Then, degranulation of neutrophils in vitro and generation of reactive intermediates by these cells under the influence of AMP extract were assessed. As estimated, the addition of AMP extract into cultures of macrophages decreased superoxide anion generation after 5 days of incubation. Furthermore, AMP extract inhibited degranulation and respiratory burst in neutrophils, therefore in this regard it suppress proinflammatory effect of two studied populations of leukocytes.
Cathelicidins ; In Vitro Techniques ; Intercellular Signaling Peptides and Proteins ; Leukocytes ; Macrophages ; Methods ; Neutrophils* ; Peptides ; Platelet-Rich Plasma* ; Respiratory Burst ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Superoxides

The objective of this study was to construct an improved thioredoxin fusion protein expression system, and express the cathelicidin-derived peptide, Lf-CATH2. The improved fusion vector Lf-CATH2-pET32α(-TS) was successfully constructed by firstly deleting the thrombin site and S tag from the pET-32α vector, then inserting the Lf-CATH2 plus a thrombin site instead. Afterwards, Lf-CATH2 was expressed in Escherichia coli as fusion protein. After the cleavage by thrombin, Lf-CATH2 was released and subsequently separated using affinity chromatography. The antimicrobial activity of purified Lf-CATH2 was also examined. The improved expression vector significantly increased enzyme cleavage efficiency by 37%, and Lf-CATH2 could be expressed in high yield and maintain the biological activity. This novel thioredoxin fusion protein expression system enables a quick production of high-yield bioactive cationic peptides like cathelicidins.
Cathelicidins ; biosynthesis ; Chromatography, Affinity ; Escherichia coli ; Genetic Vectors ; Recombinant Fusion Proteins ; biosynthesis ; Thioredoxins ; genetics

OBJECTIVETo investigate the effect of antibacterial peptide LL-37 on the integrity of Acinetobacter baumannii biofilm.

METHODSA model of Acinetobacter baumannii biofilm in vitro was constructed by plates and crystal violet staining method, and the minimal inhibitory concentration of LL-37 was determined by broth dilution. The biofilm morphology was observed by scanning electron microscopy and biofilm formation was analyzed by the crystal violet staining of the adherent biofilm in the presence of antibacterial peptide LL-37.

RESULTSIn the Acinetobacter baumannii biofilm model, the minimal inhibitory concentration of LL-37 was 64 µg/ml; LL-37 caused structural damage of the biofilm at a low concentration of 2.5 µg/ml. The biofilm was decreased gradually as the concentration of LL-37 increased.

CONCLUSIONLL-37 even at a concentration far below its minimal inhibitory concentration can cause structural damage of Acinetobacter baumannii biofilm in vitro.

Acinetobacter baumannii ; drug effects ; physiology ; Biofilms ; drug effects ; Cathelicidins ; pharmacology ; Microbial Sensitivity Tests

The purpose of this review is to explain the function of LL-37 in the pathogenesis of chronic sinusitis. LL-37 is the only human cathelicidin identified so far. LL-37 is an integral part of the innate immune,the role of which in chronic sinusitis is attracting more and more s attention.
Cathelicidins ; metabolism ; Chronic Disease ; Humans ; Sinusitis ; immunology ; metabolism ; pathology

OBJECTIVEThe aim of this study was to investigate the role of macrophages in promotion of ovarian tumor cell proliferation mediated by over-expression of antimicrobial peptide LL-37.

METHODSTo co-culture ovarian tumor cells SKOV3, 3AO and HO-8910 with macrophages. The Transwell(®) inserts system was used in the co-culture model. The effect of macrophages promoted ovarian tumor cell proliferation was assessed by BrdU-ELISA and cell number counting. Expressions of mRNA and protein of LL-37 in the macrophages and SKOV3 cells were determined by RT-PCR and Western blot analysis. To observe that LL-37 is responsible for macrophage-promoted ovarian tumor cells growth, LL-37 neutralizing antibody was added to abrogate the LL-37 activation.

RESULTSThe cell number assay showed that after 4 days coincubation with macrophages in the proportion of 1:0.5, the number of SKOV3 cells increased from (6.0 ± 0.5)×10(4) to (11.8 ± 1.3)×10(4), showing a significant difference (P < 0.05). It also showed that the growth of the SKOV3 cells was dependent on the macrophage number (P < 0.05). The number variability of 3AO and HO-8910 cells was as the same as SKOV3 cells upon co-culture with macrophages. As determined by BrdU-ELISA, the resulted proliferation of ovarian tumor cells was similar to the result of cell number counting. RT-PCR and Western blot results showed that the expression of LL-37 mRNA and protein in the macrophages was remarkably enhanced in a time dependent manner upon coincubation with SKOV3 cells, but did not work in SKOV3 cells. BrdU-ELISA assay exhibited that treatment of cells with LL-37 significantly stimulated HO-8910 and 3AO cell proliferation. Addition of LL-37 neutralizing antibody markedly inhibited macrophage-promoted ovarian tumor cell (SKOV3, 3AO and HO-8910 cells) proliferation. The OD values of these three cells were decreased from 2.95 ± 0.11 to 1.45 ± 0.04, from 3.39 ± 0.36 to 1.32 ± 0.09 and from 3.93 ± 0.17 to 1.68 ± 0.23, respectively (P < 0.05).

CONCLUSIONSOver-expression and release of LL-37 from macrophages is responsible for proliferation of ovarian tumor cells in co-culture condition. The data presented indicate that LL-37 may be critical for macrophage-induced tumor progression.

Antibodies, Neutralizing ; pharmacology ; Cathelicidins ; genetics ; metabolism ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Coculture Techniques ; Female ; Humans ; Macrophages ; cytology ; physiology ; Ovarian Neoplasms ; metabolism ; pathology ; RNA, Messenger ; metabolism

BACKGROUND: A stem cell is an undifferentiated cell that has the potential for self-renewal and differentiation. Adipose-derived stem cells (ADSCs) have advantages in accessibility and abundance compared to other kinds of stem cells and produce many growth factors and hormones. OBJECTIVE: We investigated whether ADSC cultured media could be used as a therapy for atopic dermatitis. METHODS: ADSC cultured media was topically applied twice daily for 5 days to oxazolone-treated atopic dermatitis-like hairless mice. RESULTS: Topical application of ADSC cultured media improved the epidermal permeability barrier and keratinocyte differentiation, and restored the predominant Th2 phenotype when compared to vehicle. ADSC cultured media-treated epidermis also showed an increase in the expression of antimicrobial peptides cathelin-related antimicrobial peptide, mouse beta-defensein 3. CONCLUSION: Topical ADSC cultured media could be useful in the treatment of atopic dermatitis.
Animals ; Cathelicidins ; Dermatitis, Atopic ; Epidermis ; Intercellular Signaling Peptides and Proteins ; Keratinocytes ; Mice ; Oxazolone ; Peptides ; Permeability ; Phenotype ; Stem Cells