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.

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

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.

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.

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)

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.

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.

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 role of heparin-binding protein, procalcitonin, C-reactive protein and white blood cell in respiratory tract bacterial infection / 中华检验医学杂志

Objective To evaluate the diagnostic value of the heparin-binding protein (HBP),procalcitonin (PCT),C-reactive protein (CRP),white blood cell (WBC) in respiratory tract bacterial infection.Methods 66 respiratory tract bacterial infection patients,37 respiratory tract non-bacterial infection patients and 39 control group in the Third Xiangya Hospital from October 2015 to March 2017 was selected as objects in this prospective study.The levels of HBP,PCT and CRP in blood of the objects were tested with ELESA,immunofluorescence assay,immunoturbidimetry respectively;WBC counts were taken by Sysmex XE-5000 blood analyzer.The difference among the three groups was analyzed by Student's t test,one-way ANOVA or Wilcoxon test.Receiver operating characteristic curve was utilized to analyze the diagnostic value of HBP,PCT,CRP and WBC in respiratory tract bacterial infection.Results The plasma level of HBP were 36.30 (7.78-89.36) ng/ml,5.57 (4.37-8.23) ng/ml,2.84 (1.53-6.51) ng/ml in respiratory tract bacterial infection group,respiratory tract non-bacterial infection group and control group respectively.The socre of PCT were 0.08 (0.04-0.83) ng/ml,0.09 (0.04-0.30) ng/ml,0.04 (0.03-0.08) ng/ml.The socre of CRP were 56.20 (19.33-76.23) mg/L,34.40 (2.15-83.95) mg/L,(2.20 ± 0.99) mg/L.The socre of WBC count were (10.59 ±4.58) × 109/L,8.40 (5.80-11.88) × 109/L,(6.14± 1.31) × 109/L.There were statistically significant differences in HBP scores between respiratory tract bacterial infection group and respiratory tract non-bacterial infection group or control group (Z =-4.828,P ＜0.001;Z =-5.685,P ＜ 0.001).There were no statistically significant differences in PCT,CRP and WBC scores between respiratory tract bacterial infection group and non-bacterial infection group (F =0.045,P ＞ 0.05;F =0.100,P ＞ 0.05;F =2.417,P ＞ 0.05),but significant differences between respiratory tract bacterial infection group and control group (Z =-2.881,P ＜ 0.05;Z =-6.595,P ＜ 0.001;t =6.499,P ＜ 0.001).The area under curve (AUC) of HBP,PCT,CRP and WBC diagnosing respiratory tract bacterial infection was 0.89,0.69,0.95 and 0.85 respectively.The AUC of HBP differential diagnosising was 0.80.Conclusion HBP can be used as an efficient supplementary indicator for respiratory tract bacterial infection,the differential diagnostic value is superior to PCT,CRP and WBC.

Detection and clinical application value of antimicrobial peptide hCAP18 in serum of colorectal carcinoma patients / 中华检验医学杂志

Objectve To evaluate the serum level of antimicrobial peptide human cationic antimicrobial protein 18 (hCAP18) in colorectal patients and it auxiliary diagnosis and prognosis value.Methods Case-control study was used.The serum level of hCAP18 was measured by enzyme linked immunosorbent assay(ELISA) in 68 cases with colorectal patients of department of gastrointestinal surgery and 40 cases healthy people of department of physical examination from January 2014 to Junc 2015 in Tongji Hosptial of Tongji University.The concentrations of hCAP18 in serum of colorectal patients before and surgery were analyzed.Immunohistochemistry was used to detect hCAP18 expression in colorectal carcinoma.The effect of hCAP18 on colon carcinoma cell proliferation was detected by BrdU-ELISA and soft agar colony formation assay.The sensitivity and specificity of serum hCAP18 for the diagnosis of eolorectal were evaluated using the receiver operating characteristic curves(ROC).Date was analyzed by using the ttest and one-way analysis of variance.Results hCAP18 serum levels in colon cancer of stage Ⅰ,Ⅱ,llⅢ and Ⅳ patients were (0.46 ± 0.18) mg/L,(0.65 ± 0.45) mg/L,(1.26 ± 0.68) mg/L and (2.35 ± 1.06)mg/L.Mean value was(1.16 ±0.88) mg/L,which was significantly higher than in normal people (0.19 ±0.07) mg/L (t =5.290,P ＜ 0.05).hCAP18 levels had significantly decreased in serum of colorectal patients after 30 d surgery compared to preoperative results [from (1.16 ± 0.88) mg/L to (0.26 ± 0.06) mg/L;t =3.971,P ＜ 0.05].Immunohistochemistry results showed hCAP18 was high expression in colon cancer tissue compared with adjacent tissues;BrdU-ELISA assay results showed HCTll6 and SW480 cell proliferation increased significantly after 0.05-1 mg/L of hCAP18 treatment;Soft agar clone formation experiment proved hCAP18 could significant enhance clone formation of HCT116 and SW480 colon cancer cell lines.The size of clonal cluster of HCT116 was increased from (145.40 ± 35.20) μm to (370.80 ± 32.65) μm (t =10.50,P ＜ 0.05) and SW480 was increased from (101.00 ± 27.10) μm to (369.00 ± 27.29) μm (t =15.58,P ＜0.05);The numbers of clonal cluster of HCT116 was increased from 8.50 ± 2.30 to 42.80 ± 6.60 (t =3.945,P ＜ 0.05) and SW480 was increased from 6.20 ± 1.70 to 46.00 ± 7.20 (t =4.775,P ＜ 0.05).ROC analysis of serum hCAP18 yielded an AUC (area under the ROC curve) of 0.93 (95％ CI =0.859-0.999)with 91.17％ sensitivity and 80.00％ specificity,which was higher than the CEA[0.78 (95％ CI =0.699-0.933)].Conclousions Detection of serum hCAP18 shows a good sensitivity and specificity for the auxiliary diagnosis of colon cancer.It is possible to be potential detection index for noninvasive diagnosis and monitoring progression of colon cancer.hCAP18 could promote the proliferation of colon cancer cells,it played an important role in the progression of colon cancer.

Comparative research of inflammatory factors in child patients with sepsis / 中华检验医学杂志

Objective To investigate clinical value of inflame factors in child patients with sepsis at different time points before the diagnosis time.Methods A retrospective model was performed in this study.24 child patients with sepsis in Department of Paediatrics from January 2014 to October 2016 were selected.At the time 72 h(group A),48 h(group B),24 h(group C) before the diagnosis time,plasma levels of HBP and serum levels of IL-6,IL-10 were detected by ELISA,and pre calcitonin (PCT) and high sensitive C reactive protein (hs-CRP) were detected by immunofluorescence.Compared to the same period,22 healthy cases were selected as the control.Repeated measure anova and Receiver operating characteristic curve analysis were performed.Results The plasma levels of HBP were (9.69 ± 1.30) μg/L,(12.82 ±2.03) μg/L,(15.46 ± 1.02) μg/L,(18.60 ± 1.10) μg/L at group A,group B,group C before the diagnosis time respectively.The plasma levels of HBP at all time points before the diagnosis time were significantly higher than the control (t =6.27,P ＜ 0.01;t =16.82,P ＜ 0.01;t =25.16,P ＜ 0.01).The serum levels of HBP at group B,group C were significantly higher than the last time point (t =5.62,P ＜0.01;t =10.25,P ＜ 0.01).Receiver operating characteristic curve(ROC) revealed that the areas of HBP at group A(0.823),group B (0.898),was significantly higher than the other inflame factors(Z =2.41,P ＜0.01;Z=2.02,P＜0.05;Z=0.38,P＞0.05;Z=0.32,P＞0.05)(Z=0.43,P＞0.05;Z=0.46,P＞0.05;Z =0.26,P ＞ 0.05;Z =0.57,P ＞ 0.05).It also revealed that at group C,area of PCT(0.941) was significantly higher than the other inflame factors (Z =0.12,P ＞ 0.05;Z =0.08,P ＞ 0.05;Z =0.03,P ＞0.05;Z-0.10,P ＞ 0.05).Conclusions HBP has a wide diagnostic window period for sepsis.IL-6,IL-10,PCT and hs-CRP have diagnostic value in partial periods of sepsis.

Comparison of the clinical value of plasma heparin-binding protein, procalcitonin and C-reactive protein in the early diagnosis of sepsis / 中华检验医学杂志

Objective To assess the clinical utility of measurement of plasma heparin-binding protein (HBP) in diagnosis and prognosis of sepsis.Methods This is a retrospective study on the performance of plasma heparin-binding protein, procalcitonin and C-reaction protein in the early diagnosis of sepsis. Thirty-one patients with sepsis, 16 patients with severe sepsis, 12 patients with septic shock and 37 control patients without confirmed sepsis, all admitted to the Intensive Care Units (ICU) of the Third Hospital Affiliated to Wenzhou Medical University and Wenzhou Central Hospital from August 2014 to November 2016, were enrolled in the study. The plasma level of HBP, procalcitonin (PCT) and C-reactive protein (CRP) were measured, and the detailed clinical data were retrieved from the patient chart records for all patients described above. Comparison of each laboratory and clinical parameters between groups was carried out by Non-parameter Test. The efficiency of each parameter was calculated by receiver operating characteristics curves (ROC) analysis. The correlation between HBP, PCT or CRP and clinical or other laboratory parameters was explored using Spearman correlation analysis.Results HBP was significantly elevated in patients with severe sepsis[(100.65±58.82)ng/ml and(31.86±36.87)ng/ml,Z=-3.856,P<0.05;(100.65±58.82)ng/ml and(24.96±17.49)ng/ml,Z=-3.556,P<0.05]and in patients with septic shock[(148.28±99.73)ng/ml and(31.86±36.87)ng/ml,Z=-4.432,P<0.05;(148.28±99.73)ng/ml and(24.96±17.49)ng/ml,Z=-4.157,P<0.05], respectively, while CRP[(154.64±62.90)mg/L and(92.56±67.49)mg/L,Z=-2.749,P<0.05;(154.64±62.90)mg/L and (79.21±51.80)mg/L,Z=-3.218,P<0.05]and PCT[(32.86±39.93)ng/ml and(2.70±6.24)ng/ml,Z=-3.395,P<0.05;(32.86±39.93)ng/ml and(4.21±14.94)ng/ml,Z=-4.092,P<0.05]were increased only in patients with septic shock (P<0.05).For HBP, the area of under the ROC curves (AUC) was the biggest (AUC=0.687), indicating the clinical significance(P<0.05) with excellent sensitivity(0.729) at the optimal cut-off value(18.58 ng/ml). In addition, HBP(APTT: r=0.244, P=0.016;PT: r=0.351, P<0.001;INR: r=0.314, P=0.002;D-Dimer: r=0.334, P=0.001;lactic acid: r=0.394, P<0.001), CRP(APTT: r=0.271, P=0.008;PT: r=0.348, P=0.001;INR: r=0.264, P=0.009;D-Dimer: r=0.257, P=0.012;lactic acid: r=0.329, P=0.001) and PCT(APTT: r=0.375, P<0.001;PT: r=0.523, P<0.001;INR: r=0.535, P<0.001;D-Dimer: r=0.254, P=0.013;lactic acid: r=0.422, P<0.001)were positively correlated to coagulation function and to lactate.Conclusion HBP could probably be acted as an important biomarker for diagnosis and prognosis for patients with sepsis, esp., for patients with severe sepsis and septic shock.

Efeito microbiológico e citotóxico de sistemas nanoestruturados bioadesivos contendo fragmentos de peptídeos / Microbiological and citotoxicity effect of nanostructured bioadhesive system containing peptide fragments

O uso de agentes antimicrobianos naturais que reduzam a adesão e proliferação de S. mutans no biofilme poderia ser uma estratégia interessante para o controle da cárie dentária. No entanto, a estabilidade química e física de alguns desses agentes, como os peptídeos catiônicos antimicrobianos e fragmentos de peptídeos, pode ser comprometida por fatores externos, como temperatura e pH, reduzindo sua ação antimicrobiana. Com isso, os objetivos deste estudo foram desenvolver e caracterizar sistemas de liberação de fármaco nanoestruturados bioadesivos para a incorporação dos fragmentos peptídicos D1-23 e P1025 e avaliar seu efeito citotóxico e atividade contra biofilme de S. mutans. A primeira formulação (F1), composta de ácido oleico, polyoxypropylene-(5)-polyoxyethylene-(20)-cetyl alcohol (PPCA), Carbopol® 974P e Carbopol® 971P, foi analisada por microscopia de luz polarizada (MLP), reologia e bioadesão in vitro. A concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) de D1-23 foram determinadas contra S. mutans para posterior avaliação da atividade sobre biofilme formado após 4h e 24h de tratamento. A segunda formulação (F2) foi selecionada a partir de três diferentes concentrações de ácido oleico, PPCA e Carbopol® 974P. Cada formulação foi analisada por MLP, espalhamento de raios x a baixo ângulo (SAXS), reologia e bioadesão. CIM e CBM de P1025 sobre S. mutans e seu efeito quando incorporado ou não em F2 sobre biofilme de S. mutans em formação foram analisados. A citotoxicidade em células epiteliais HaCat foi avaliada para os dois sistemas líquido cristalino (SLC) usando testes de MTT. Análise descritiva foi realizada para os dados dos ensaios de caracterização e para os ensaios microbiológicos e citotóxicos os dados foram submetidos aos testes de ANOVA/Tukey ou Kruskall-Wallis/Mann-Whitney U (p<0.05). Os resultados indicaram que F1 apresentou características de SLC com alta viscosidade e bioadesão. CIM e CBM de D1-23 foram de 15,60 e 31,25µg/mL, respectivamente. D1-23 incorporado em F1 apresentou melhores resultados contra biofilme de S. mutans que quando em solução, após 24h de tratamento. F2 apresentou melhores propriedades reológicas e força bioadesiva comparada aos demais sistemas, caracterizando um SLC. P1025 teve somente efeito inibitório sobre S. mutans (CIM=0.25 mg/mL). O efeito antibiofilme de P1025 incorporado em F2 foi observado após 24h de tratamento, principalmente quando aplicado na fase de adesão. Ambos os SLC contendo D1-23 e P1025 não apresentaram toxicidade sobre as células epiteliais, nas condições de tempo e concentrações avaliadas. A incorporação de peptídeos em SLC bioadesivos nanoestruturados aumenta suas propriedades antimicrobianas, podendo ser uma interessante estratégia para a prevenção da cárie dentária(AU)

The use of natural antimicrobial agents for reducing the adhesion and proliferation of S. mutans in the biofilm could be an interesting strategy for the control of dental caries. However, the chemical and physical stability of some natural antimicrobials, such as cationic antimicrobial peptides and peptide fragments, can be compromised by external factors such as temperature and pH, reducing their antimicrobial action. Thus, the objectives of this study were to develop and characterize nanostructured bioadhesive drug delivery systems for the incorporation of D1-23 and P1025 peptide fragments and to evaluate their citotoxicy and activity against S. mutans biofilm. The first formulation (F1) was composed of oleic acid, polyoxypropylene- (5) -polyoxyethylene- (20) - cetyl alcohol (PPCA), Carbopol® 974P and Carbopol® 971P and analyzed by polarized light microscopy (PLM), rheology and in vitro bioadhesion. Minimum inhibitory concentration (MIC) and minimal bacterial concentration (MBC) of D1-23 were determined against S. mutans for further evaluation of activity against S. mutans biofilm after 4h and 24h of treatment. The second formulation was selected from three different concentrations of oleic acid, PPCA and Carbopol® 974P. Each formulation was analyzed by PLM, small-angle x-ray scattering (SAXS), rheology and bioadhesion. MIC and MBC of P1025 were determined against S. mutans. Thus, P1025 was incorporated in the best formulation (F2). The effect of P1025 incorporated or not into F2 on S. mutans biofilm formation was analyzed. Cytotoxicity in HaCat epithelial cells for both formulations was evaluated using MTT assays. Descriptive analysis was performed for the characterization assays and data from microbiological and cytotoxic assays were submitted to ANOVA / Tukey or Kruskall-Wallis / Mann-Whitney U (p<0.05). The results indicated that F1 presented characteristics of liquid-crystalline type system (LCS) with high viscosity and bioadhesion. The MIC and MBC of D1-23 were 15.60 and 31.25µg / mL, respectively. D1-23 incorporated in F1 showed better results than D1-23 in solution against S. mutans biofilm after 24h. F2 had better rheological properties and bioadhesive strength compared to other systems analyzed and characteristics of LCS. P1025 had only inhibitory effect against S. mutans (MIC=0.25mg/mL). The antibiofilm effect of P1025 incorporated into F2 was observed after 24h of treatment, mainly when applied in surface-bound salivary phase. Both LCS had no toxicity on epithelial cells, considering time and concentrations tested. The incorporation of peptides in nanostructured bioadhesive LCS increased their antimicrobial properties and could be an interesting strategy for caries prevention(AU)

Skin barrier in atopic dermatitis: beyond filaggrin

Abstract: Atopic dermatitis is a chronic inflammatory skin disease with a complex pathogenesis, where changes in skin barrier and imbalance of the immune system are relevant factors. The skin forms a mechanic and immune barrier, regulating water loss from the internal to the external environment, and protecting the individual from external aggressions, such as microorganisms, ultraviolet radiation and physical trauma. Main components of the skin barrier are located in the outer layers of the epidermis (such as filaggrin), the proteins that form the tight junction (TJ) and components of the innate immune system. Recent data involving skin barrier reveal new information regarding its structure and its role in the mechanic-immunological defense; atopic dermatitis (AD) is an example of a disease related to dysfunctions associated with this complex.

Análise do perfil fenotípico e funcional das células natural Killer e linfócitos TCD8+ no Líquen plano / Analysis of phenotypic and functional profile of Natural Killer cells and CD8 + T lymphocytes in Lichen planus

Líquen plano (LP) é uma doença mucocutânea de natureza inflamatória crônica de etiologia ainda desconhecida. Alterações na resposta imune inata, como aos padrões moleculares associados à patógenos (PAMPs) e padrões moleculares associados ao dano (DAMPs) podem levar à inflamação crônica e contribuir com a patogênese do LP. OBJETIVO: Avaliar o efeito da ativação via o DAMP S100A8 e o receptor Toll-like 4 (TLR-4) em células Natural killer (NK) e TCD8 citotóxicas e suas subpopulações de memória/efetoras em pacientes com LP. MÉTODOS: Foram selecionados 25 pacientes com LP (22 mulheres, 3 homens) com idade média de 43,46 anos ± 8,46 e um grupo controle com 25 indivíduos (22 mulheres, 3 homens) com idade média de 42 anos ± 5,5. A determinação transcricional e da expressão por imunohistoquimica dos DAMPs S100A8, HMGB-1 e de TLR-4 e RAGE foi realizada em biópsias de lesões cutâneas de indivíduos com LP, e os níveis séricos de S100A8, HMGB-1, MICA e MICB foram determinados por ELISA. As células mononucleares (CMNs) de sangue periférico foram avaliadas por citometria de fluxo quanto a frequência de TNF, IL-1beta e o marcador de desgranulação CD107a em células TCD8+ e células NK CD56+ e suas subpopulações. A avaliação da via de sinalização de TLR em células TCD8+ purificadas e ativadas com S100A8 foi analisada por PCR array e a determinação da expressão de mRNA dos componentes do inflamassoma em células TCD8+ ativadas com S100A8 por PCR em tempo real. RESULTADOS: Foi evidenciado nos indivíduos com LP elevada expressão da proteína S100A8 nas lesões cutâneas e de HMGB-1, TLR-4 e RAGE na derme, em paralelo ao aumento da expressão de mRNAs para S100A8 e S100A9 e diminuição de RAGE. Além disto, uma elevação dos níveis séricos do dímero S100A8/A9 foi detectada nos pacientes comparados aos controles, ao contrário do DAMP HMGB-1 que mostrou níveis similares em ambos os grupos. A influência do S100A8 em células TCD8+ e células NK, foi analisada em CMNs pela ativação...

Lichen planus (LP) is a mucocutaneous inflammatory chronic disease of unknown etiology. Alterations in the innate immune response such as the pathogen-associated molecular pattern (PAMPs) and damage-associated molecular pattern (DAMPs) can lead to chronic inflammation and contribute to the pathogenesis of LP. OBJECTIVE: Evaluate the effect of the activation trough the DAMP S100A8 and the Toll-like receptor 4 (TLR-4) on the Natural killer cells (NK) and cytotoxic TCD8 cells and their memory / effector subsets in LP disease. METHODS: We selected 25 patients with LP (22 women, 3 men) with a mean age of 43.46 years ± 8.46 and a control group of 25 subjects (22 women, 3 men) with a mean age of 42 ± 5, 5. The transcriptional determination and protein expression by immunohistochemistry of DAMPs, S100A8 and HMGB-1 as well as TLR-4 and RAGE was performed on biopsies of skin lesions from patients with LP, and serum levels of S100A8, HMGB-1, MICA and MICB were determined by ELISA. Peripheral blood mononuclear cells (PBMCs) were assessed by flow cytometry to evaluate the frequency of TNF, IL-1beta and the degranulation marker CD107a in CD8+ T cells and CD56 + NK cells and their subsets. The evaluation of the TLR signaling pathway in purified CD8 + T cells activated with S100A8 were analyzed by PCR array and the determination of mRNA expression of inflammasome components on CD8 + T cells activated by S100A8 was measured by real time PCR. RESULTS: It was shown in the LP individuals an increased expression of the S100A8 protein in the cutaneous lesions and HMGB-1, TLR-4 and RAGE in the dermis, in parallel to increased level of mRNAs for S100A8 and S100A9 and decreased expression of RAGE. Moerover, increased serum levels of the dimer S100A8 / A9 was detected in patients compared to controls, in contrast to DAMP HMGB1 that revealed similar levels in both groups. The influence of S100A8 in CD8 + T cells and NK cells, was analyzed in PBMC activating with...

Diseño de péptidos basado en la secuencia análoga al represor negativo icaR de Staphylococcus sp / Design of peptides based on similar sequence of negative repressor icaR of Staphylococcus sp / Desenho de peptídeos com base na sequência similar ao repressor negativo icaR dos Staphylococcus sp

La biopelícula como un mecanismo de virulencia en Staphylococcus involucrada en infecciones intrahospitalarias es regulada por un represor negativo icaR, responsable de la transcripción completa del operón icaADBC. La búsqueda de dominios funcionales por modulación computacional de icaR permitió hallar las secuencias peptídicas con actividad biológica análoga a la proteína icaR. Mediante biología computacional se diseñaron péptidos empleando el programa de predicción AntiBP (http://www.imtech.res.in/raghava/antibp/); la síntesis química se hizo por Nα-Fmoc y se caracterizaron y purificaron tres moléculas por RP-HPLC y MALDI-TOF. Se evaluó su seguridad biológica mediante ensayo de actividad citotóxica realizada sobre macrófagos murinos de la línea J774 y la actividad hemolítica se determinó mediante el uso de glóbulos rojos. Los tres péptidos caracterizados IR1, IR2 e IR3, presentaron estructura secundaria predominantemente alfa helicoidal, alto grado de pureza y alto score antimicrobiano; además, mostraron baja toxicidad, evidenciada por la actividad citotóxica y hemolítica en las concentraciones ensayadas y en comparación con los controles usados, que permitiría su potencial uso como moléculas candidatas o principios activos con actividad análoga al represor nativo icaR, frente a la biopelícula de los Staphylococcus sp.

Staphylococcus sp. biofilm, formed as a mechanism of virulence that is involved in hospital acquired infections, is regulated by a negative repressor icaR, which is responsible for the full transcription of the operon icaADBC. This study, through functional commands by computational modulation of icaR, allowed to find peptide sequences with similar biological activity to the icaR protein. Peptides were designed by means of computational biology using the prediction program AntiBP (http://www.imtech.res.in/raghava/antibp/). The chemical synthesis of peptides was performed by Nα-Fmoc. The purification and characterization of three molecules were carried out using RP-HPLC and MALDI-TOF. Biological safety of peptides was evaluated by tests of cytotoxic activity on murine macrophage cells line J774, and their hemolytic activity was determined by using red cells. The three characterized peptides IR1, IR2 and IR3 presented a predominantly secondary alpha helical structure with a high degree of purity and high antimicrobial scores. In addition, the peptides exhibited low toxicity, proved by their low cytotoxic and hemolytic activity in the tested concentrations and in comparison to the standards used. These results allow the potential use of these peptides as candidate molecules or active principles with similar activity to the native repressor icaR against the Staphylococcus biofilm.

O biofilme formado como um mecanismo de virulência em Staphylococcus sp., que está envolvido com infecções intra-hospitalares, é regulado por um repressor negativo icaR, o qual é responsável pela plena transcrição do operão icaADBC. Por tanto, o presente estudo, avaliando a segurança biológica de moléculas, concebeu peptídeos antibiofilme semelhantes ao repressor icaR. Por meio da biologia computacional foram concebidos peptídeos usando o programa de predição AntiBP (http://www.imtech.res.in/raghava/antibp/) para identificar as sequências com uma atividade biologicamente similar à da proteína icaR. A Síntese química dos peptídeos se fez pelo Nα-Fmoc e foram caracterizadas e purificadas três moléculas por RP-HPLC e MALDI-TOF. O ensaio de atividade citotóxica foi realizado nos macrófagos murinos da linha J774 e a atividade hemolítica foi determinada por meio do uso de glóbulos vermelhos. Foram caraterizados três peptídeos IR1, IR2 e IR3, além de mostrarem uma estrutura secundaria predominantemente alfa helicoidal, com alto grau de pureza, alto score antimicrobiano e baixa toxicidade, estes podem ser postulados como moléculas candidatas ou princípios ativos com uma atividade similar ao repressor nativo icaR frente ao biofilme dos Staphylococcus sp.

ß-defensin-2 in breast milk displays a broad antimicrobial activity against pathogenic bacteria / ß-defensina 2 no leite materno mostra uma ampla atividade antimicrobiana contra bactérias patogênicas

OBJECTIVE: To describe the antimicrobial activity of ß-defensin-2 produced in the mammary gland and secreted in human breast milk. METHODS: The peptide production was performed by DNA cloning. ß-defensin-2 levels were quantified in 61 colostrum samples and 39 mature milk samples from healthy donors, by an indirect enzyme-linked immunosorbent assay (ELISA). Using halo inhibition assay, this study assessed activity against seven clinical isolates from diarrheal feces of children between 0 and 2 years of age. The activity of ß-defensin-2 against three opportunistic pathogens that can cause nosocomial infections was determined by microdilution test. RESULTS: The peptide levels were higher in colostrum (n = 61) than in mature milk samples (n = 39), as follows: median and range, 8.52 (2.6-16.3) µg/ml versus 0.97 (0.22-3.78), p < 0.0001; Mann-Whitney test. The recombinant peptide obtained showed high antimicrobial activity against a broad range of pathogenic bacteria. Its antibacterial activity was demonstrated in a disk containing between 1-4 µg, which produced inhibition zones ranging from 18 to 30 mm against three isolates of Salmonella spp. and four of E. coli. ß-defensin-2 showed minimum inhibitory concentrations (MICs) of 0.25 µg/mL and 0.5 µg/mL for S. marcescen and P. aeruginosa, respectively, while a higher MIC (4 µg/mL) was obtained against an isolated of multidrug-resistant strain of A. baumannii. CONCLUSIONS: To the authors' knowledge, this study is the first to report ß-defensin-2 levels in Latin American women. The production and the activity of ß-defensin-2 in breast milk prove its importance as a defense molecule for intestinal health in pediatric patients. .

OBJETIVO: Descrever a atividade antimicrobiana da defensina-beta 2 na glândula mamária e secretada no leite materno humano. MÉTODOS: A produção de peptídeos foi realizada por clonagem de DNA. Os níveis de defensina-beta 2 foram quantificados em 61 amostras de colostro e 39 de leite maduro de doadoras saudáveis pelo teste ELISA indireto. Por um ensaio de halo de inibição, avaliamos a atividade contra sete isolados clínicos diarreicos de crianças entre 0 e 2 anos. A atividade da defensina 2 contra três patógenos oportunistas que podem causar infecções nosocomiais foi determinada pelo teste de microdiluição. RESULTADOS: Os níveis de peptídeos estavam significativamente maiores nas amostras de colostro (n = 61) que de leite maduro (n = 39), como segue: 8,52 (2,6-16,3 µg/mL) mediana e faixa em comparação a 0,97 (0,22-3,78), p < 0,0001; teste de Mann-Whitney. O peptídeo recombinante foi obtido da alta atividade antimicrobiana demonstrada contra uma ampla gama de bactérias patogênicas. Sua atividade antibacteriana foi demonstrada em um disco contendo entre 1-4 µg, que produziu zonas de inibição entre 18 e 30 mm contra três isolados de Salmonella spp. e quatro de E. coli. A defensina-beta 2 demonstrou concentrações inibitórias mínimas (CIMs) de 0,25 µg/mL e 0,5 µg/mL para S. marcescen and P. aeruginosa, ao passo que uma CIM maior (4 µg/mL) foi obtida contra um isolado de cepa multirresistente de A. baumannii. CONCLUSÕES: Até onde sabemos, este estudo é o primeiro a relatar níveis de defensina em mulheres da América Latina. A produção e a atividade da defensina 2 no leite materno comprovam sua importância como uma molécula de defesa para a saúde intestinal em pacientes pediátricos. .

Fatores microbiológicos e imunológicos envolvidos no desenvolvimento da cárie precoce da infância / Microbiological and immunological factors for the development of early childhood caries

A cárie precoce da infância (CPI) é ainda um grave problema de saúde pública no mundo, principalmente em países em desenvolvimento. Estudos têm sugerido a associação da ingestão frequente de carboidratos fermentáveis como a sacarose, altas contagens de microrganismos cariogênicos e maior vulnerabilidade imunológica da criança na etiologia da CPI. O objetivo deste estudo foi avaliar os aspectos microbiológicos e imunológicos associados ao desenvolvimento da cárie precoce da infância. Crianças com idade entre 36 e 60 meses foram selecionadas e divididas em três grupos: LC - livres de cárie, CPI e CPI-S (CPI-severa). Questionário sobre os aspectos socioeconômico-culturais, hábitos de higiene bucal e diários de dieta foram respondidos pelos responsáveis. Foram coletadas amostras de saliva e biofilme dental das crianças e processadas para subsequentes avaliações laboratoriais. Em seguida, os níveis de IgA salivar total e contra GbpB de S. mutans foram determinados por ELISA e Western blot, respectivamente, as concentrações salivares dos peptídeos catiônicos antimicrobianos (PCAM): defensinas hBD-2 e hBD-3, catelicidina LL-37 e histatina 5 (HTN-5) por ELISA e a presença e os níveis salivares de Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. e Scardovia wiggsiae por qRT-PCR, sendo que estes dados foram correlacionados com os níveis salivares e no biofilme dental de estreptococos mutans (SM) e Lactobacillus spp. por meio de cultivo em meios específicos. Os resultados mostraram que as crianças com CPI-S apresentaram menor renda familiar quando comparadas às crianças LC ou CPI. Contudo, a ingestão de açúcar não diferiu entre os grupos. O grupo CPI-S apresentou maior contagem de SM na saliva/biofilme em relação aos grupos LC e CPI. Houve uma correlação positiva entre a resposta de IgA contra GbpB e os níveis de SM, quando a população geral foi avaliada. Quando apenas crianças com altos níveis de SM foram comparadas, o grupo CPI-S...

Early childhood caries (ECC) is still a serious public health problem worldwide, especially in developing countries. Studies have been suggested the association among frequent intake of fermentable carbohydrates such as sucrose, high cariogenic microorganism’s counts and child’s immune vulnerability in the etiology of ECC. The objective of this study was to evaluate the microbiological and immunological factors for the development of early childhood caries. 36 to 60 monthold children were selected and distributed into three groups: caries free (CF), ECC and S-ECC (severe-ECC). Questionnaires about socio-economic-cultural data, oral hygiene habits and food-frequency diary were completed by the parents. Saliva and dental biofilm were collected from children and processed for subsequent laboratorial tests. The following analyses were determined: total IgA and IgA response against S. mutans GbpB by ELISA and Western blot, respectively; salivary concentrations of antimicrobial peptides (AMPs): defensins hBD-2 and hBD-3, cathelicidin LL-37 and histatin 5 (HTN-5) by ELISA; salivary detection and quantification of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. and Scardovia wiggsiae by qRT-PCR, and these data were correlated with mutans streptococci (MS) and Lactobacillus spp. levels by culture in specific medium. Results showed that S-ECC children had reduced family income compared to ECC and CF. However, sugar intake did not differ among the groups. S-ECC group had higher MS count than CF/ECC groups. Positive correlations between salivary IgA response against GbpB and MS counts were found when the entire population was evaluated. When children with high mutans streptococci counts were compared, S-ECC group showed a significant decrease in IgA antibody levels against GbpB compared to CF group. This finding was not observed for ECC group. The present study showed positive correlations between salivary hBD-2 and HTN-5 with…

Fatores microbiológicos e imunológicos envolvidos no desenvolvimento da cárie precoce da infância / Microbiological and immunological factors for the development of early childhood caries

A cárie precoce da infância (CPI) é ainda um grave problema de saúde pública no mundo, principalmente em países em desenvolvimento. Estudos têm sugerido a associação da ingestão frequente de carboidratos fermentáveis como a sacarose, altas contagens de microrganismos cariogênicos e maior vulnerabilidade imunológica da criança na etiologia da CPI. O objetivo deste estudo foi avaliar os aspectos microbiológicos e imunológicos associados ao desenvolvimento da cárie precoce da infância. Crianças com idade entre 36 e 60 meses foram selecionadas e divididas em três grupos: LC - livres de cárie, CPI e CPI-S (CPI-severa). Questionário sobre os aspectos socioeconômico-culturais, hábitos de higiene bucal e diários de dieta foram respondidos pelos responsáveis. Foram coletadas amostras de saliva e biofilme dental das crianças e processadas para subsequentes avaliações laboratoriais. Em seguida, os níveis de IgA salivar total e contra GbpB de S. mutans foram determinados por ELISA e Western blot, respectivamente, as concentrações salivares dos peptídeos catiônicos antimicrobianos (PCAM): defensinas hBD-2 e hBD-3, catelicidina LL-37 e histatina 5 (HTN-5) por ELISA e a presença e os níveis salivares de Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. e Scardovia wiggsiae por qRT-PCR, sendo que estes dados foram correlacionados com os níveis salivares e no biofilme dental de estreptococos mutans (SM) e Lactobacillus spp. por meio de cultivo em meios específicos. Os resultados mostraram que as crianças com CPI-S apresentaram menor renda familiar quando comparadas às crianças LC ou CPI. Contudo, a ingestão de açúcar não diferiu entre os grupos. O grupo CPI-S apresentou maior contagem de SM na saliva/biofilme em relação aos grupos LC e CPI. Houve uma correlação positiva entre a resposta de IgA contra GbpB e os níveis de SM, quando a população geral foi avaliada. Quando apenas crianças com altos níveis de SM foram comparadas, o grupo CPI-S...

Early childhood caries (ECC) is still a serious public health problem worldwide, especially in developing countries. Studies have been suggested the association among frequent intake of fermentable carbohydrates such as sucrose, high cariogenic microorganism’s counts and child’s immune vulnerability in the etiology of ECC. The objective of this study was to evaluate the microbiological and immunological factors for the development of early childhood caries. 36 to 60 monthold children were selected and distributed into three groups: caries free (CF), ECC and S-ECC (severe-ECC). Questionnaires about socio-economic-cultural data, oral hygiene habits and food-frequency diary were completed by the parents. Saliva and dental biofilm were collected from children and processed for subsequent laboratorial tests. The following analyses were determined: total IgA and IgA response against S. mutans GbpB by ELISA and Western blot, respectively; salivary concentrations of antimicrobial peptides (AMPs): defensins hBD-2 and hBD-3, cathelicidin LL-37 and histatin 5 (HTN-5) by ELISA; salivary detection and quantification of Streptococcus mutans, Streptococcus sobrinus, Lactobacillus spp., Bifidobacterium spp. and Scardovia wiggsiae by qRT-PCR, and these data were correlated with mutans streptococci (MS) and Lactobacillus spp. levels by culture in specific medium. Results showed that S-ECC children had reduced family income compared to ECC and CF. However, sugar intake did not differ among the groups. S-ECC group had higher MS count than CF/ECC groups. Positive correlations between salivary IgA response against GbpB and MS counts were found when the entire population was evaluated. When children with high mutans streptococci counts were compared, S-ECC group showed a significant decrease in IgA antibody levels against GbpB compared to CF group. This finding was not observed for ECC group. The present study showed positive correlations between salivary hBD-2 and HTN-5 with…