In vitro efficacy and combined drug sensitivity of lycosin-I against methicillin-resistant Staphylococcus aureus / 中华检验医学杂志

Objective:The aim of this study was to evaluate the in vitro activity of lysosin-Ⅰ against Methicillin-resistant Staphylococcus aureus (MRSA) and its synergistic effect with eight common antibacterial drugs against MRSA. Methods:This study was conducted following the design principles of a randomized controlled trials. Ten MRSA isolates, clinically isolated from the Second Xiangya Hospital of Central South University between September and November 2021, were determined the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and bactericidal kinetic test of lycosin-Ⅰ in vitro anti-MRSA by micro-broth dilution method. Additionally, the micro-broth chessboard dilution method was utilized to evaluate the in vitro efficacy of lycosin-Ⅰ in combination with eight common antimicrobial agants, including penicillin, erythromycin, levofloxacin, gentamicin, rifampicin, minocycline, vancomycin, and linezolid. Results:The MIC range of lycosin-Ⅰ against MRSA was found to be between 4-8 mg/L with the MIC 50 and MIC 90 were 4 mg/L and 8 mg/L, respectively. The range of MBC was also between 4-8 mg/L, and the ratio of MBC/MIC was 1-2. The bactericidal kinetics test revealed that the number of surviving MRSA clinical isolates and standard strains initially decreased rapidly but then showed a resurgence when the concentration of lycosin-Ⅰ was 1/2 MIC or MIC. While, the bacterial load gradually reduced until complete elimination when the concentration was at 2 MIC or 4 MIC. The combination of lycosin-Ⅰ and gentamicin exhibited mainly synergistic effects, while the combination with other antibiotics showed mainly additive effects. Moreover, the combination of lycosin-Ⅰ and antibacterial drugs can significantly reduce the MIC 50 and MIC 90 of antibiotics. Conclusion:lycosin-Ⅰ has great antibacterial and bactericidal activity against MRSA in vitro with rapid and thorough sterilization effect and it can play a synergistic or additive role when combined with other antibacterial drugs against MRSA in vitro.

Combined Treatment with Fluoride and Antimicrobial Peptide GH12 Efficiently Controls Caries in vitro and in vivo.

Combining fluoride and antimicrobial agents enhances regulation of acid and exopolysaccharide production by biofilms. The combination also weakens the acidogenic and aciduric bacteria that contribute to caries, achieving stronger caries-controlling effects with lower concentrations of fluoride. In previous studies, antimicrobial peptide GH12 has been shown to inhibit lactic acid and exopolysaccharide synthesis in various cariogenic biofilm models, and reduce the proportion of acidogenic bacteria and Keyes caries scores in a rat caries model. The current study aimed to elucidate the effect of a combination of low concentrations of sodium fluoride (NaF) and GH12 and to determine the mechanism by which GH12/NaF combination controls caries. The GH12/NaF combination contained 8 mg/L GH12 and 250 ppm NaF. A rat caries model was built, and rat dental plaque was sampled and cultivated on bovine enamel slabs in vitro and subjected to short-term treatment (5 min, 3 times/day). The caries-controlling effects were evaluated using Keyes scoring and transverse microradiography. The results showed that the GH12/NaF combination significantly decreased the onset and development of dental caries, as well as mineral content loss and lesion depth in vitro (p < 0.05). For the caries-controlling mechanisms, 16S rRNA sequencing of in vivo dental plaque revealed that populations of commensal bacteria Rothia spp. and Streptococcus parasanguinis increased in the GH12/NaF group. In contrast, Veillonella, Lactobacillus, and Streptococcus mutans decreased. Furthermore, the GH12/NaF combination significantly reduced biomass, lactic acid, and exopolysaccharides production of in vitro biofilm (p < 0.05). Overall, fluoride and GH12 efficiently arrested caries development and demineralization by regulating the microbiota and suppressing acid and exopolysaccharide production in biofilms.

An Overview of Databases and Bioinformatics Tools for Plant Antimicrobial Peptides.

Antimicrobial Peptides (AMPs) are small, ribosomally synthesized proteins found in nearly all forms of life. In plants, AMPs play a central role in plant defense due to their distinct physicochemical properties. Due to their broad-spectrum antimicrobial activity and rapid killing action, plant AMPs have become important candidates for the development of new drugs to control plant and animal pathogens that are resistant to multiple drugs. Further research is required to explore the potential uses of these natural compounds. Computational strategies have been increasingly used to understand key aspects of antimicrobial peptides. These strategies will help to minimize the time and cost of "wet-lab" experimentation. Researchers have developed various tools and databases to provide updated information on AMPs. However, despite the increased availability of antimicrobial peptide resources in biological databases, finding AMPs from plants can still be a difficult task. The number of plant AMP sequences in current databases is still small and yet often redundant. To facilitate further characterization of plant AMPs, we have summarized information on the location, distribution, and annotations of plant AMPs available in the most relevant databases for AMPs research. We also mapped and categorized the bioinformatics tools available in these databases. We expect that this will allow researchers to advance in the discovery and development of new plant AMPs with potent biological properties. We hope to provide insights to further expand the application of AMPs in the fields of biotechnology, pharmacy, and agriculture.

The effects of Peptide Mel4-coated titanium plates on infection rabbits after internal fixation of open fractures.

INTRODUCTION: Infection after internal fixation surgery is an orthopedic serious complication which affect the fracture healing. The primary objective of this study was to verify the effects of a Peptide Mel4-coated titanium plate applied in the treatment of infection after internal fixation of open fracture. MATERIALS AND METHODS: Eighty-eight rabbits were intravenously inoculated with Staphylococcus aureus or Pseudomonas aeruginosa suspensions. Bacterial cultures were obtained from titanium plates at 1st, 3rd, 5th, 7th and 9th days. Blood samples were collected at 1st, 3rd, 5th, 7th and 9th days after the infection. RESULTS: Mel4-coated titanium plates have significant inhibitory effects on Staphylococcus aureus and Pseudomonas aeruginosa (P < 0.05), and there are significant differences in serum IL-1 and TNF-α levels (P < 0.05). CONCLUSION: We suggest that the use of Mel4-coated titanium plates may be a promising way to control postoperative infection of open fracture in vivo.

Calcium-based antimicrobial peptide compounds cooling gel for the treatment of atopic dermatitis: a randomized, double-blind controlled clinical study / 中华皮肤科杂志

Objective:To evaluate clinical efficacy and safety of calcium-based antimicrobial peptide compounds cooling gel (CAPCS cooling gel) in the treatment of atopic dermatitis (AD) .Methods:A randomized, double-blind, active-controlled clinical study was conducted. From July 2019 to May 2020, 80 adult patients with mild-to-moderate AD were enrolled from Beijing Friendship Hospital, Capital Medical University, and randomly and equally divided into 2 groups: test group topically treated with CAPCS cooling gel, control group topically treated with hydrocortisone cream, and the treatment was performed thrice a day for 4 consecutive weeks. Before, 1, 2 and 4 weeks after the start of treatment, efficacy was evaluated according to the Eczema Area and Severity Index (EASI), Visual Analog Scale (VAS), and Investigator′s Global Assessment (IGA) scores, and adverse events were recorded. Efficacy and safety were compared by using repeated measures analysis of variance and chi-square test.Results:Of the 80 patients with AD, 43 were males and 37 were females, and the age was 52.71 ± 16.71 years. Before the treatment, there was no significant difference in gender, age, EASI, VAS or IGA scores between the two groups (all P > 0.05). After 1- and 2-week treatment, there was no significant difference in the response rate between the test group (10.00% [4/40], 57.50% [23/40], respectively) and control group (15.00% [6/40], 52.50% [21/40] respectively, both P > 0.05). After 4-week treatment, the response rate was significantly higher in the test group (82.50%, 33/40) than in the control group (57.50%, 23/40, P < 0.05). Compared with the control group, the test group showed significantly decreased VAS scores after 1-, 2- and 4-week treatment ( U = 1253.00, 1121.00, 1091.50, respectively, all P < 0.05). No drug-related adverse events were observed in either of the groups. Conclusion:CAPCS cooling gel is safe and effective in the treatment of mild-to-moderate AD in adults, and can be applied in clinic.

Host defense peptides clavanins A and MO reduce in vitro osteoclastogenesis

Aim: Several systemic diseases, such as periodontitis and apical periodontitis, can cause extensive bone resorption. Host defense peptides may have the potential for the development of novel therapies for the bone resorption process. This study evaluated the potential of host defense peptides clavanins A, MO, and LL-37 in in vitro osteoclastogenesis. Methods: RAW 264.7 cultures were stimulated with recombinant of receptor activator of nuclear factor kappa B ligand in the presence of different tested concentrations of host defense peptides, besides calcium hydroxide and doxycycline. Cellular viability, nitric oxide production, and a number of differentiated osteoclast-like cells were also evaluated. Results: Results showed that none of the substances were cytotoxic, except for 128 µg.mL-1 of doxycycline after 3 days. Host defense peptides, calcium hydroxide, and doxycycline did not interfere in nitric oxide production or downregulated it. An exception was observed in the presence of 2 µg.mL-1 of doxycycline, in which nitric oxide production was up-regulated. All host defense peptides were capable of reducing osteoclast-like cell differentiation. Conclusion: Host defense peptides clavanins A and MO demonstrated to be potential suppressors of osteoclastogenesis in vitro without interfering in cellular viability and nitric oxide production. These promising results need to be further analyzed in in vivo models of bone resorption

Combined Training Improves the Expression Profile of Inflammation-associated Antimicrobial Peptides, MicroRNAs, and TLR-4 in Patients with Multiple Sclerosis.

Some antimicrobial peptides (AMPs), microRNAs (miRs), and Toll-like receptor 4 (TLR-4) are involved in autoimmune diseases, which may be affected by exercise training. The purpose of this study was to investigate the effect of an eight-week combined exercise training (aerobic and resistance) on the expression of inflammatory factors, including, human beta-defensin-2 (hBD-2), cathelicidin (LL-37), TLR-4, miR-23b, miR-155, and miR-326 in women with relapsing and remitting multiple sclerosis (RRMS), which has not been investigated yet. Twenty-three women (20-40 years) with RRMS were randomized into the combined training (CT) and control (CON) groups. The CT group subjects completed eight weeks of supervised CT using a treadmill and stationary bicycle for aerobic exercise and weight machines for resistance exercise. The expression levels of hBD-2, LL-37, TLR-4, miR-23b, miR-155, and miR-326 were measured by real-time polymerase chain reaction (RT-PCR) at the baseline and end of the study. Although the expression of hBD-2 and miR-23b decreased in both CT and CON groups, the reduction was lower in the CT group than in the CON group (p=0.001). The expression of LL-37 in the CT group remained unchanged, but that of the CON group increased; thus, the between-group difference was significant. Although the TLR-4, miR-155, and miR-326 expression increased in both groups compared to the baseline, the increase in the CT group was lower than the CON group. Our results showed that the combined training might improve inflammatory symptoms by affecting the expression of some AMPs, miRs, and TLR-4 in patients with relapsing and remitting multiple sclerosis.

Bronchoalveolar Lavage and Plasma Cathelicidin Response to 25-Hydroxy Vitamin D Supplementation: A Pilot Study.

INTRODUCTION: Cathelicidin is a vitamin D-regulated, antimicrobial peptide involved in the innate immune response of the airways. Reduced plasma cathelicidin concentrations are independently associated with worse pulmonary outcomes in current and former smokers. This study aimed to determine whether oral vitamin D supplementation in vitamin D-deficient current smokers increases plasma and bronchoalveolar lavage (BAL) cathelicidin levels. METHODS: Vitamin D-deficient (25-hydroxy vitamin D [25-OH vitamin D] <20 ng/ml) smokers (n=17) underwent collection of plasma and BAL for cathelicidin and 25-OH vitamin D measurements before and after 8 weeks of oral supplementation with 50,000 IU vitamin D3 weekly. Differences between baseline and 8-week levels of cathelicidin and 25-OH vitamin D in blood and BAL were assessed along with correlations between serum 25-OH vitamin D, plasma cathelicidin, and BAL cathelicidin. RESULTS: At baseline, there was no correlation between BAL and plasma cathelicidin. There was a significant increase in 25-OH vitamin D (median 17.0 to 43.3 ng/mL, p<0.001) after 8 weeks of vitamin D supplementation. There was no change in plasma cathelicidin (p=0.86), BAL cathelicidin (p=0.31), or BAL 25-OH vitamin D (p=0.89). There was no correlation between serum 25-OH vitamin D and either BAL or plasma cathelicidin post-supplementation. CONCLUSIONS: Oral vitamin D supplementation, while increasing serum 25-OH vitamin D levels, does not increase plasma or BAL cathelicidin levels in vitamin D-deficient, active smokers. The lack of increased BAL cathelicidin may be explained by multiple factors related to dosing, smoking effects, or putative mechanisms of engagement. Future studies are needed to determine the effects of vitamin D supplementation on lung and blood functional activity.

Predicted regulatory SNPs reveal potential drug targets and novel companion diagnostics in psoriasis.

Psoriasis is an autoimmune disease associated with interleukins, their receptors, key transcription factors and more recently, antimicrobial peptides (AMPs). Cathelicidin LL-37 is an AMP proposed to play a fundamental role in psoriasis etiology. With our proprietary software SNPClinic v.1.0, we analyzed 203 common SNPs (MAF frequency â€‹> â€‹1%) in proximal promoters of 22 genes associated with psoriasis. These include nine genes which protein products are classic drug targets for psoriasis (TNF, IL17A, IL17B, IL17C, IL17F, IL17RA, IL12A, IL12B and IL23A). SNPClinic predictions were run with DNAseI-HUP chromatin accessibility data in eight psoriasis/epithelia-relevant cell lines from ENCODE including keratinocytes (NHEK), TH1 and TH17 lymphocytes. Results were ranked quantitatively by transcriptional relevance according to our novel Functional Impact Factor (FIF) parameter. We found six rSNPs in five genes (CAMP/cathelicidin, S100A7/psoriasin, IL17C, IL17RA and TNF) and each was confirmed as true rSNP in at least one public eQTL database including GTEx portal and ENCODE (Phase 3). Predicted regulatory SNPs in cathelicidin, IL17C and IL17RA genes may explain hyperproliferation of keratinocytes. Predicted rSNPs in psoriasin, IL17C and cathelicidin may contribute to activation and polarization of lymphocytes. Predicted rSNPs in TNF gene are concordant with the epithelium-mesenchymal transition. In spite that these results must be validated in vitro and in vivo with a functional genomics approach, we propose FOXP2, RUNX2, NR2F1, ELF1 and HESX1 transcription factors (those with the highest FIF on each gene) as novel drug targets for psoriasis. Furthermore, four out of six rSNPs uncovered by SNPClinic v.1.0 software, could also be validated in the clinic as companion diagnostics/pharmacogenetics assays for psoriasis prescribed drugs that block TNF-α (e.g. Etanercept), IL-17 (e.g. Secukinumab) and IL-17 receptor (Brodalumab).

Effect of Dermaseptin S4 on C. albicans Growth and EAP1 and HWP1 Gene Expression.

Increasing resistance and changes in the spectrum of Candida infections have generated considerable interest in the development of new antifungal molecules. The use of antimicrobial peptides (AMPs) appears to be a promising approach. Frog skin AMPs (such as dermaseptins) have shown antimicrobial activity against several pathogens. In this study, we aimed to test the antimicrobial efficacy of dermaseptin S4 (DS4) against C. albicans. We determined the minimal inhibitory concentration (MIC) of DS4, and investigated the effects of the DS4 at low concentrations on human primary gingival fibroblasts. Additionally, we evaluated the effect of DS4 on C. albicans growth, form changes, and biofilm formation, as well as the expression of certain virulent genes. Our data show that DS4 completely inhibits C. albicans growth at a concentration of 32 µg/mL referring to the MIC of DS4. It should be noted that even with low concentrations (below 16 µg/mL), DS4 still have significant growth reduction of C. albicans, but were not toxic to human gingival fibroblasts. DS4 inhibited the transition from yeast to hyphae, and decreased the biofilm formation by reducing the biofilm mass weight. Surface morphological changes in the yeast cell membrane were observed following exposure to DS4. The gene expression analyses revealed that DS4 significantly decreased the expression of EAP1 and HWP1 genes. Overall results suggest the potential use of DS4 as an antifungal therapy to prevent C. albicans pathogenesis.

A Bi-Functional Targeted P28-NRC Chimeric Protein with Enhanced Cytotoxic Effects on Breast Cancer Cell Lines.

One of the emerging therapeutic strategies for targeted therapy of cancer is the use of chimeric proteins. The p28 peptide has the ability of selective entrance and activating apoptosis in breast cancer cells. The NRC antimicrobial peptide showed cytotoxic activity on various breast cancer cell lines including drug-resistant cells and also on normal cells. Here we designed a chimeric protein consisting of these peptides to determine their targeted effects and to enhance their cytotoxic effects on breast cancer cells. The chimeric protein was cytotoxic to MDA-MB-231 and MCF7 breast cancer cell lines in a dose-dependent manner after 48 h of treatment. In addition, the cytotoxic effects of the p28 alone were significantly lower than the chimeric protein indicating the additive or enhanced effects of the two peptides. Flow cytometry analysis showed that the induced cell death is mediated via apoptosis. The designed chimeric protein had enhanced effects on breast cancer cell lines and exerted its anticancer effects on MCF7 breast cancer cells through mitochondrial caspase dependent and -independent apoptotic pathways. Taken together, the results of this study suggested the chimeric protein to be a reasonable anti-cancer agent which must be further evaluated by subsequent in-vitro and in-vivo preclinical studies.

Diagnositic value of urine heparin-binding protein, interleukin-6 and white blood cell in bacterial urinary tract infection / 中华检验医学杂志

Objective To evaluate the diagnostic values of urinary heparin-binding protein (HBP), interleukin-6 (IL-6) and white blood cell (WBC) levels in bacterial urinary tract infection (UTI). Methods A case-control method was used. Urine of 157 cases of bacterial UTI, 61 cases of non-infection, and 40 cases of normal controls were collected in the Third Xiangya Hospital of Central South University from September 2017 to March 2018. U-HBP levels were measured in duplicate using a commercial HBP ELISA, U-IL-6 concentrations were analyzed with an up-conversion luminescence. The method of quantitative culture of bacteria was used to identify pathogenic species. Rapid dipstick tests and urinary sediment analyses were detected by FUS-2000 Urinalysis Hybrid. For continuous variables with skewed distributions, comparisons among the three groups were performed using the nonparametric Kruskal-Wallis test, and Mann-Whitney U test was used to further evaluate the difference between two groups. The Chi-square test was applied to analyze dichotomous. Receiver operating characteristic curve (ROC curve) was constructed to analyze the clinical diagnostic values of U-HBP, U-IL-6 and U-WBC for bacterial UTI. Results The levels of U-HBP in UTI group, non-UTI group and control group were 513.43 (50.45-644.40) ng/ml, 55.65 (20.43-314.55) ng/ml and 4.83 (3.28-12.63) ng/ml. The scores of U-IL-6 were 5.72 (3.84-9.02) pg/ml, 5.31 (4.31-6.39) pg/ml and 5.06 (4.56-6.18) pg/ml. The scores of U-WBC were 205 (24-754) cells/μl, 34 (13-117) cells/μl and 0 (0-0) cell/μl. There were statistically significant differences of U-HBP and U-WBC among the three groups (HU-HBP=83.192, HU-WBC=100.416, P<0.05), but no significant difference for U-IL-6 (HU-IL-6=2.585, P>0.05). The best Youden indexes of U-HBP and U-WBC diagnosing bacterial UTI were 0.475 and 0.441, respectively. The best cut-off level of U-HBP and U-WBC was 64.35 ng/ml and 119.25 cells/μl, respectively. Conclusions Testing the level of U-HBP was important for auxiliary diagnosing bacterial UTI, but testing U-IL-6 wasn't.

Clinical value of determining heparin-binding protein of cerebrospinal fluid in children with purulent meningitis / 中华检验医学杂志

Objective@#To explore the clinical value of determining heparin-binding protein(HBP) of cerebrospinal fluid(CSF) in the diagnosis and prognostic prediction in children with purulent meningitis(PM).@*Methods@#76 children with PM, 55 children with viral encephalitis(VE) and 40 control children with non-infectious diseases, all admitted to Hunan Children′ Hospital from August 2018 to January 2019, were enrolled in this retrospective study. Children with PM were divided into favorable prognosis group and poor prognosis group according to the Glasgow Outcome Scale on discharge. CSF HBP, white blood cell count(WBC), percentage of neutrophilic granulocyte(N%), glucose(Glu), total protein(TP), lactic dehydrogenase (LDH) and serum procalcitonin(PCT) were analyzed on the first day of admission(DAY1) in PM group, VE group and control group, and on the seventh day of admission(DAY7) in PM group. Nonparametric tests were used to detect the differences of the laboratory indexes and Spearman rank correlation test was utilized to analyze the correlation between HBP and other markers. Receiver operating characteristic curves (ROC curves) were established to evaluate the values of the detection indexes in the diagnosis and prognosis of PM.@*Results@#The differences of CSF HBP[63.09(18.10-272.19)ng/mL, 5.90(5.90-6.40)ng/mL and 5.90(5.90-5.90)ng/mL], WBC[365.00(20.00-1285.00)×106/L, 21.00(8.00-30.00)×106/L and 13.50(7.25-21.00)×106/L], N%[0.65(0.50-0.79), 0.19(0.10-0.25) and 0.21(0.15-0.27)], Glu[1.97(1.07-3.08)mmol/L, 2.89(2.66-3.42)mmol/L and 3.04(2.68-3.42)mmol/L], TP[1.43(0.63-1.88)g/L, 0.23(0.16-0.32)g/L and 0.13(0.10-0.31)g/L], LDH[152.00(46.50-461.50)IU/L, 16.00(13.20-22.00)IU/L and 16.00(10.25-19.75) IU/L] and serum PCT[1.35(0.19-9.33)ng/mL, 0.06(0.03-0.11)ng/mL and 0.08(0.05-0.14)ng/mL] levels on DAY1 were statistically significant among PM group, VE group and control group(HHBP=138.62, HWBC=69.72, HN%=106.67, HGlu=34.08, HTP=68.00, HLDH=85.11, HPCT=79.20, P<0.001). HBP had the largest area under curve(AUC=0.997) for the diagnosis of PM, and had excellent sensitivity, specificity, positive predictive value, negative predictive value (98.70%, 97.90%, 97.40%, 98.94%, respectively) at the optimal cut-off value (11.84 ng/mL). Compared with DAY1,CSF HBP, WBC, N%, TP, LDH and serum PCT levels on DAY7 were statistically lower in favorable prognosis group(P<0.05). The differences for all the indexes between DAY1 and DAY7 in poor prognosis group were not statistically significant, however. It was not significant for all the indexes on DAY1 to predict poor prognosis(P>0.05). But the indexes on DAY7 for predicting poor prognosis were significant (P<0.05) and HBP still had the largest AUC (0.976) for predicting the poor prognosis with good sensitivity, specificity, positive predictive value, negative predictive value (100.0%, 93.8%, 76.3%, 100.0%, respectively) at the optimal cut-off value(128.84 ng/mL). CSF HBP was positively correlated to CSF WBC, N%, TP, LDH, serum PCT level(rWBC=0.670, rN%=0.802, rTP=0.562, rLDH=0.524, rPCT=0.436, P<0.001) and negatively correlated to CSF Glu level(r=-0.469, P<0.001).@*Conclusions@#CSF HBP is valuable in the diagnosis and prognostic prediction in children with purulent meningitis.

Efeito isolado ou combinado de flavonoides e peptídeos catiônicos sobre biofilme endodôntico e sua influência na viabilidade celular, capacidade de migração e inibição de citocinas em fibroblastos / Effect of flavonoids and cationic peptides, alone or in combination, on endodontic biofilm and their influence on cell viability, migration capacity and inhibition of cytokines in fibroblasts

Este trabalho foi dividido em dois capítulos que objetivou avaliar: 1) o efeito isolado ou combinado do flavonoide epigallocatechin-3-gallate (EGCG) em associação com o peptídeo LL-37 e seu análogo KR-12-a5 sobre a viabilidade celular de fibroblastos e sobre cultura planctônica, biofilme simples, dual-espécies e túbulos dentináios e 2) as interações sinérgicas do EGCG e proantocianidina do oxicoco (A-type cranberry proanthocyanidins, AC-PAC), quando usado em combinação com LL-37 ou KR-12-a5 sobre a viabilidade celular, a capacidade de migração e inibição das citocinas em cultura de fibroblastos (HGF-1), quando estimuladas ou não pelo lipopolissacarídeo de A. actinomycetencomitans (LPS). No capítulo 1, a concentração inibitória mínima (MIC), a concentração bactericida mínima (MBC) e concentração inibitória fracionária (FIC) de EGCG, LL-37 e KR-12-a5 foram determinadas a partir de valores decrescentes dos compostos por meio dos métodos de microdiluição e checkerboard contra Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii e Fusobacterium nucleatum após 24 horas de tratamento. Fibroblastos da linhagem L-929 foram expostos a combinações de EGCG com peptídeos em diferentes concentrações e o metabolismo celular avaliado por ensaios de MTT. Os compostos com melhor efeito antimicrobiano e citotóxico foram avaliados por 24-36h, isoladamente ou em combinação, em biofilmes individuais ou biofilmes de dual-espécies com E. faecalis formados em placas de poliestireno por 48h por meio de contagem bacteriana. Os biofilmes de E. faecalis também foram cultivados em túbulos dentinários por 2 semanas, tratados com EGCG, KR-12-a5 e EGCG + KR-12-a5 e a porcentagem de células mortas foi determinada pela análise de imagens usando Microscopia Confocal. No capítulo 2, a linhagem celular de fibroblastos gengivais humanos primários HGF-1 foi pré-tratada durante 2 h com EGCG ou AC-PAC a 25 e 12,5 µg / mL, LL-37 ou KR-12-a5 a 0,06 e 0,03 µM ou com uma combinação de EGCG + ACPAC; AC-PAC + KR-12-a5; AC-PAC + LL-37; EGCG + KR-12-a5 ou EGCG + LL-37, nas mesmas concentrações. As culturas celulares foram então estimuladas com 50 µg/mL de LPS por 24-48h. A viabilidade celular e migração foram analisadas usando ensaios colorimétricos e fluorescentes, respectivamente. A quantificação de citocinas foi determinada por ensaios multiplex ELISA. Os resultados mostraram que em condições planctônicas, EGCG + KR-12-a5 apresentaram efeito sinérgico ou aditivo contra todas as bactérias testadas, com FIC menor que os valores de MIC obtidos pelos compostos isolados. As combinações de EGCG e peptídeos testados não foram tóxicas para os fibroblastos, uma vez que o crescimento celular foi superior a 70%. Em condições de biofilme simples, EGCG + KR-12-a5 eliminou S. mutans e A. israelii e reduziu E. faecalis e F. nucleatum. Para biofilmes de duas espécies, quando E. faecalis foi combinado com S. mutans, EGCG + KR-12-a5 teve efeito sinérgico eliminando S. mutans e reduzindo estatisticamente as contagens de E. faecalis. Em biofilmes associando E. faecalis e A. israelii ou F. nucleatum, EGCG + KR-12-a5 eliminaram E. faecalis e promoveram redução de A. israelii e F. nucleatum, embora não tenha sido observada diferença estatística entre os compostos. EGCG + KR-12-a5 reduziu mais de 80% dos biofilmes de E. faecalis nos túbulos dentinários. Dentre os grupos experimentais estudados, o EGCG, principalmente a 25 e 12,5 µg/mL estimulou o crescimento de fibroblastos, protegendo-os dos efeitos do LPS. Efeito sinérgico entre EGCG + AC-PAC, EGCG + LL-37 e EGCG + KR-12-a5 no metabolismo celular também foi observado na presença de LPS. Combinações do EGCG com AC-PAC ou KR-12-a5 e AC-PAC com LL-37 foram capazes de aumentar estatisticamente a migração celular. EGCG, AC-PAC, LL-37 e KR-12-a5 promoveram a redução de citocinas individualmente ou em combinação (EGCG + AC-PAC e EGCG + KR12-a5) mais especificamente para IL-6, IL-8, GM- CSF e TNF-α. Conclui-se que a associação de EGCG e KR-12-a5 é citocompatível e promove um efeito sinérgico contra bactérias associadas a infecções endodônticas, sob condições planctônicas e de biofilme. O EGCG, isoladamente ou associado ao AC-PAC e ao KR-12-a5, aumenta a viabilidade e migração celular, bem como a inibição de citocinas por fibroblastos estimulados por LPS. A associação de EGCG com KR-12-a5 poderia ser uma opção de princípio ativo em medicações para fins endodônticos(AU)

This study was divided in two chapters that aimed to evaluate: 1) the effect of flavonoid epigallocatechin-3-gallate (EGCG), cationic peptide LL-37 peptide and its analogue KR12-a5, alone or in combination, on fibroblast cell viability and on bacteria in planktonic and single/dual-species biofilms/dentin tubules; 2) the synergistic interactions of EGCG and cranberry proanthocyanidins (A-type cranberry proanthocyanidins, AC-PAC), when used in combination with LL-37 or KR-12-a5 on cell viability, the ability to induce cell migration and inhibit cytokines in culture of fibroblasts (HGF-1) when stimulated or not by the lipopolysaccharide of A. actinomycetencomitans (LPS). For the chapter 1, Minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and fractional inhibitory concentration (FIC) of EGCG, LL-37 and KR-12-a5 were determined from decreasing values of the compounds by Streptococcus mutans, Enterococcus faecalis, Actinomyces israelii and Fusobacterium nucleatum against microdilution and checkerboard after 24 hours of treatment. L-929 fibroblasts were exposed to combinations of EGCG with peptides at different concentrations and cell metabolism assessed by MTT assays. The compounds if the best antimicrobial and cytotoxic effect were also evaluated for 24-36h, alone or in combination, in 48h singleor dual-species biofilms with E. faecalis formed on polystyrene plates by bacterial counting. E. faecalis biofilms were also cultured in dentin tubules for 2 weeks and treated with EGCG, KR-12-a5 and EGCG + KR-12-a5 to determine the percentage of dead cells by analysis of images using Confocal Microscopy. For the chaper 2, primary human gingival fibroblast HGF-1 cell line was pretreated for 2 h with either EGCG or AC-PAC at 25 and 12.5 µg/mL, LL-37 or KR-12-a5 at 0.03 and 0.06 µM or with a combination of EGCG + AC-PAC; AC-PAC + KR-12-a5; AC-PAC + LL-37; EGCG + KR-12-a5 or EGCG + LL37, at the same concentrations. Cell cultures were then stimulated with 50 µg/mL LPS for 24-48h. Cell viability and migration were analyzed using colorimetric and fluorescent assays, respectively. Quantification of cytokines was determined by multiplex ELISA assays. The results show that in planktonic conditions, EGCG + KR-12- a5 showed a synergistic or additive effect against all the bacteria tested, with FIC lower than the MIC values obtained by the compounds alone. Combinations of EGCG and peptides tested were not toxic to fibroblasts, since cell growth was higher than 70%. Under single biofilm conditions, EGCG + KR-12-a5 eliminated S. mutans and A. israelii and reduced E. faecalis and F. nucleatum. For dual- species biofilms, when E. faecalis was combined with S. mutans, EGCG + KR-12-a5 had a synergistic effect by eliminating S. mutans and statistically reducing E. faecalis counts. In biofilms associated with E. faecalis and A. israelii or F. nucleatum, EGCG + KR-12-a5 eliminated E. faecalis and promoted reduction of A. israelii and F. nucleatum, although no statistical difference was observed between the compounds. EGCG + KR-12-a5 reduced more than 80% of the E. faecalis biofilms in the dentin tubules. Among the experimental groups studied, EGCG, mainly at 25 and 12.5 µg/mL stimulated the growth of fibroblasts, protecting them from the effects of LPS. Synergistic effect between EGCG + AC-PAC, EGCG + LL-37 and EGCG + KR12-a5 on cell metabolism was also observed in the presence of LPS. Combinations of EGCG with AC-PAC or KR-12-a5 and AC-PAC with LL-37 were able to increase statistically cell migration. EGCG, AC-PAC, LL-37 and KR-12-α5 promoted cytokine reduction individually or in combination (EGCG + AC-PAC and EGCG + KR-12-a5) more specifically for IL-6, IL-8, GM-CSF and TNF-α. The association of EGCG and KR-12-a5 was cytocompatible and promoted a synergistic effect against bacteria associated with endodontic infections under planktonic and biofilm conditions. EGCG, alone or in combination with AC-PAC and KR-12-a5, increases cell viability and migration, as well as inhibition of cytokines by LPS-stimulated fibroblasts. The association of EGCG with KR12-a5 could be an option as active principle for medications to be used for endodontic purposes(AU)

Antimicrobial peptide GH12 suppresses cariogenic virulence factors of Streptococcus mutans.

Cariogenic virulence factors of Streptococcus mutans include acidogenicity, aciduricity, and extracellular polysaccharides (EPS) synthesis. The de novo designed antimicrobial peptide GH12 has shown bactericidal effects on S. mutans, but its interaction with virulence and regulatory systems of S. mutans remains to be elucidated. The objectives were to investigate the effects of GH12 on virulence factors of S. mutans, and further explore the function mechanisms at enzymatic and transcriptional levels. To avoid decrease in bacterial viability, we limited GH12 to subinhibitory levels. We evaluated effects of GH12 on acidogenicity of S. mutans by pH drop, lactic acid measurement and lactate dehydrogenase (LDH) assay, on aciduricity through survival rate at pH 5.0 and F1F0-ATPase assay, and on EPS synthesis using quantitative measurement, morphology observation, vertical distribution analyses and biomass calculation. Afterwards, we conducted quantitative real-time PCR to acquire the expression profile of related genes. GH12 at 1/2 MIC (4 mg/L) inhibited acid production, survival rate, EPS synthesis, and biofilm formation. The enzymatic activity of LDH and F1F0-ATPase was inhibited, and ldh, gtfBCD, vicR, liaR, and comDE genes were significantly downregulated. In conclusion, GH12 inhibited virulence factors of S. mutans, through reducing the activity of related enzymes, downregulating virulence genes, and inactivating specific regulatory systems.

Transcriptomic and Proteomic Profiling Revealed High Proportions of Odorant Binding and Antimicrobial Defense Proteins in Olfactory Tissues of the House Mouse.

Mammalian olfaction depends on chemosensory neurons of the main olfactory epithelia (MOE), and/or of the accessory olfactory epithelia in the vomeronasal organ (VNO). Thus, we have generated the VNO and MOE transcriptomes and the nasal cavity proteome of the house mouse, Mus musculus musculus. Both transcriptomes had low levels of sexual dimorphisms, while the soluble proteome of the nasal cavity revealed high levels of sexual dimorphism similar to that previously reported in tears and saliva. Due to low levels of sexual dimorphism in the olfactory receptors in MOE and VNO, the sex-specific sensing seems less likely to be dependent on receptor repertoires. However, olfaction may also depend on a continuous removal of background compounds from the sites of detection. Odorant binding proteins (OBPs) are thought to be involved in this process and in our study Obp transcripts were most expressed along other lipocalins (e.g., Lcn13, Lcn14) and antimicrobial proteins. At the level of proteome, OBPs were highly abundant with only few being sexually dimorphic. We have, however, detected the major urinary proteins MUP4 and MUP5 in males and females and the male-biased central/group-B MUPs that were thought to be abundant mainly in the urine. The exocrine gland-secreted peptides ESP1 and ESP22 were male-biased but not male-specific in the nose. For the first time, we demonstrate that the expression of nasal lipocalins correlates with antimicrobial proteins thus suggesting that their individual variation may be linked to evolvable mechanisms that regulate natural microbiota and pathogens that regularly enter the body along the 'eyes-nose-oral cavity' axis.

The differential diagnostic value of pleural effusion heparin-binding protein in parapneumonic effusion / 中华检验医学杂志

Objective The aim of this study was to evaluate the value of pleural effusion heparin-binding protein ( HBP) in differential diagnosis of parapneumonic effusion .Methods Case-control study. The pleural effusion of 189 patients with pleural effusion admitted to Quzhou People's Hospital from February to July 2018, including parapneumonic effusion (n=72), tuberculous pleural effusion (n=24), cases of malignant pleural effusion ( n=46 ) and transudative pleural effusion ( n=47 ) were collected.Routine analysis,lactate dehydrogenase(LDH),adenosine deaminase (ADA) and total protein(TP)examination of all pleural effusions were performed .The levels of heparin-binding protein in the patients'pleural fluid were measured by ELISA.The difference in the overall level of each group was determined by One-way ANOVA or LSD test followed by Kruskal-Wallis H test dependence on the homogeneity of variances .The categorical data was analyzed by chi-square test.Receiver operating characteristic ( ROC) curve was plotted to evaluate the diagnostic value of heparin-binding protein for parapneumonic effusion . Results The concentration of heparin-binding protein was low in malignant pleural effusion [15.2(8.4, 33.3) ng/ml] and transudative effusion[14.1(6.5, 23.0)ng/ml], but high in parapneumonic effusion[316.1(99.5,399.8)ng/ml]and tuberculous pleurisy [64.7 (18.6, 96.8) ng/ml] .The heparin-binding protein level in parapneumonic effusion was significantly different from the other three groups (H=120.3,P<0.05).The receiver operating characteristic curve analysis for an optimal discrimination between parapneumonic effusion from non -parapneumonic effusion could be performed at a cut-off point of 64.2 ng/ml with area under the curve of 0.953[sensitivity:88.9％(64/72), specificity:89.7％(105/117),positive predictive value:84.2％(64/76), negative predictive value:92.9％(105/113)].Conclusions Heparin-binding proteinin pleural fluid is effective to be used to classify parapneumonic effusion samples .The detection of heparin-binding protein in pleural effusion has good sensitivity and specificity .It could be a biomarker for differential diagnosis of parapneumonic effusion .

The clinical value for plasma heparin-binding protein in the early diagnosis of the neonatal sepsis / 中华检验医学杂志

Objective To investigate the clinical value of plasmatic heparin-binding protein in early diagnosis and severity gradation of neonatal sepsis.Methods Thirty-nine patients with general sepsis,37 patients with severe sepsis and 16 patients with septic shock were recruited as corresponding study groups respectively,who all had been admitted to the Neonatal Intensive Care Units(NICU)of Hunan Children′s Hospital from December 2016 to August 2017,meanwhile,34 patients with local infection and 35 patients with non infection were enrolled as relevant control group respectively who all had been admitted to each neonatal ward in the retrospective study.The level of the heparin-binding protein(HBP), procalcitonin (PCT)and high sensitive C-reactive protein(hs-CRP)of all patients were detected respectively at the beginning of hospitalization.The difference of each group was compared by use of nonparametric statistics and the efficacy of every index on diagnosis of infection and sepsis was assessed with the receiver operating characteristic curve(ROC).Results The level of HBP in sepsis group,severe sepsis group and septic shock group HBP(H=91.764,P<0.01), PCT(H=51.757,P<0.01)and hs-CRP(H=28.418,P<0.01)are significantly higher than those in local infection group and non infection group;Plasmic HBP levels of severe sepsis group[52.35(33.65,88.15)(ng/ml)]and septic shock group[73.55(60.61,145.51)(ng/ml)]are statistically higher than general sepsis group[34.12(23.04,41.79)(ng/ml)](H=24.092, P<0.01).There are no statistically differences of serum PCT and hs-CRP among these three groups[(HPCT=1.909,Hhs-CRP=0.292),P>0.05].The area under the curve(AUC)of HBP in diagnosis of neonatal sepsis and infection are 0.885 and 0.904 respectively,more higher than PCT and hs-CRP;With the cut off value of 19.8 ng/ml,the sensitivity and specificity of HBP on diagnosis of infection are 85.7%and 82.9%respectively;the sensitivity and specificity 80.4% and 88.4% for neonatal sepsis with the cut-off value of 28.0 ng/ml respectively.Conclusion HBP probably has the better clinical value than PCT and hs-CRP in the early diagnosis and severity gradation of neonatal sepsis.

Diagnostic value of plasma heparin-binding protein and pentraxin 3 in neonatal bacterial infection diseases / 中华检验医学杂志

Objective To evaluate the diagnosis value of heparin-binding protein ( HBP ) and pentraxin 3 ( PTX3 ) in neonatal bacterial infectious diseases . Methods A retrospective study was conducted on 30 septic neonatal as neonatal sepsis group and 84 local infection neonatal as general infection group from May to November 2017 in Renmin Hospital of Wuhan University .It also selected 50 high bilirubin hematic disease but without infection or shock neonatal ( control group ) .A total of 114 neonatal bacterial infection ( neonatal sepsis group and general infection group ) were divided into shock group ( 39 cases) and non-shock group ( 75 cases ) . The levels of plasma HBP and PTX3 were tested with immunoturbidimetry and ELSIA respectively .The results of procalcitonin ( PCT ) and white blood cells (WBC) counts were collected.Non-parametric test were performed for non-normal distribution data; the diagnostic performances of data were evaluated by receiver operating characteristic ( ROC) curve; pearson correlation coefficient was performed for correlation analysis .Results Plasma levels of HBP in neonatal sepsis group, general infection group and control group were (64.41 ±78.51) ng/ml, (47.16 ±50.59) ng/ml and (31.97 ±20.76) ng/ml, respectively; plasma levels of PTX3 were (2.23 ±1.44) ng/ml, (1.76 ±0.94) ng/ml and (1.26 ±0.66) ng/ml, respectively;serum levels of PCT were (31.92 ±36.65) ng/ml,( 7.72 ±9.28 ) ng/ml and ( 1.87 ±5.02 ) ng/ml, respectively.The levels of PTX3 and PCT in neonatal sepsis group were significantly higher than in general infection group (Z=3.74, Z=5.01, all P<0.05) and control group (Z=3.98, Z=5.20, all P<0.05).The levels of HBP in neonatal sepsis group were significantly higher than in control group ( Z =2.37, P <0.05 ), but there were no significant difference in neonatal sepsis group and general infection group (Z=1.16, P>0.05).The levels of PTX3 and PCT in shock group were significantly higher than in non-shock group ( Z=2.20, Z=3.70, all P<0.05), but there were no significant difference in plasma HBP of shock and non-shock group ( Z=0.37, P>0.05).The area under curve (AUC) of HBP, PTX3 and PCT were 0.683, 0.802 and 0.869 respectively in the diagnosis of neonatal bacterial infection diseases .The biggest AUC of combined diagnosis of HBP, PTX3 and PCT was 0.910.There was a positive correlation between PTX 3 and PCT ( r=0.242, P<0.05) .Conclusions PTX3 and PCT could probably be acted as an important biomarker for diagnosis of neonatal bacterial infection diseases , and combined diagnosis of HBP , PTX3 and PCT could be superior to single biomarker diagnosis.

Differential Ability of Bovine Antimicrobial Cathelicidins to Mediate Nucleic Acid Sensing by Epithelial Cells.

Cathelicidins encompass a family of cationic peptides characterized by antimicrobial activity and other functions, such as the ability to enhance the sensing of nucleic acids by the innate immune system. The present study aimed to investigate the ability of the bovine cathelicidins indolicidin, bactenecin (Bac)1, Bac5, bovine myeloid antimicrobial peptide (BMAP)-27, BMAP-28, and BMAP-34 to inhibit the growth of bacteria and to enhance the sensing of nucleic acid by the host's immune system. BMAP-27 was the most effective at killing Staphylococcus aureus, Streptococcus uberis, and Escherichia coli, and this was dependent on its amphipathic structure and cationic charge. Although most cathelicidins possessed DNA complexing activity, only the alpha-helical BMAP cathelicidins and the cysteine-rich disulfide-bridged Bac1 were able to enhance the sensing of nucleic acids by primary epithelial cells. We also compared these responses with those mediated by neutrophils. Activation of neutrophils with phorbol myristate acetate resulted in degranulation and release of cathelicidins as well as bactericidal activity in the supernatants. However, only supernatants from unstimulated neutrophils were able to promote nucleic acid sensing in epithelial cells. Collectively, the present data support a role for certain bovine cathelicidins in helping the innate immune system to sense nucleic acids. The latter effect is observed at concentrations clearly below those required for direct antimicrobial functions. These findings are relevant in development of future strategies to promote protection at mucosal surfaces against pathogen invasion.