The Association Between Serum Vitamin D Levels and Serum Cathelicidin Antimicrobial Peptide (CAMP) Levels Among Tuberculosis Patients in Comparison with Control Subjects

Objectives: To compare the serum vitamin D and serum cathelicidin antimicrobial peptide (CAMP) levels among tuberculosis patients and healthy subjects and to determine the association between serum vitamin D and cathelicidin in tuberculosis. Materials and Methods: This is a case-control study carried out at the pulmonary medicine and pediatrics departments of a tertiary care hospital in Chengalpattu. The study included 180 tuberculosis cases and 90 control subjects of both sexes between the age group of 1 to 80 years. Serum was used to estimate vitamin D and CAMP. The study was analyzed using SPSS version 21 (IBM Corp. Armonk, New York). The results were evaluated using the chi-square test at a 95% confidence interval, and P value <.05 was considered highly significant. Results: This study observed vitamin D deficiency, vitamin D insufficiency, and optimum vitamin D among 55%, 41%, and 3.89% tuberculosis cases, respectively. Similarly, vitamin D deficiency, vitamin D insufficiency, and optimum vitamin D were seen in 40%, 50%, and 10% healthy controls, respectively. Conclusion: This study found no association between serum vitamin D and serum CAMP levels in tuberculosis patients and healthy controls.

Cathelicidin LL-37 level in presence and absence of vitamin D in cultured macrophages isolated from elderly women

@#Introduction: Vitamin D deficiency and frequent infections are the two common worldwide phenomenon among elderly. Recent studies have demonstrated that vitamin D regulates the expression of specific endogenous antimicrobial peptides like cathelicidin LL-37 of macrophages and neutrophils, which is active against a broad spectrum of infectious agents. Therefore, the objective of the present study was to determine the level of cathelicidin LL-37 in macrophages of elderly women (classified according to serum 25(OH)D level) after exposure to Vibrio cholera infection and to find out the effect of 1,25(OH)2D added in vitro. Methods: This study was conducted among 40 randomly selected rural elderly women aged between 60 to 70 years of age. Their vitamin D status was assessed by the estimation of serum 25(OH)D and classified into three groups viz. sufficient (14 members), insufficient (13 members), and deficient (13 members). Later, their peripheral blood mononuclear cells (PBMC) were isolated and cultured from fresh blood. 1,25(OH)2D supplementation was given selectively at a dose of 10 ×10-8 M for 72 hours in the culture media; then exposed to infection and screened according to the objectives of this study. Results: Macrophages in all groups, except vitamin D deficient group, responded significantly in terms of LL-37 release during exposure to Vibrio cholera infection. Considering in vitro 1,25(OH)2D, supplementation responded significantly (p<0.05) in all three groups. Conclusion: Vitamin D can be used as a prophylaxis to enhance cathelicidin LL-37 release for all three groups as in the present study.

The level of antimicrobial peptides in gingival crevicular fluid and its correlation with periodontal clinical indexes in elderly patients with type 2 diabetic periodontitis / 口腔疾病防治

Objective@#To investigate the changes and significance of human beta-defensin-2 (HBD-2) and LL-37 in the gingival crevicular fluid of patients with periodontitis and type 2 diabetes mellitus (T2DM).@*Methods@#This study was conducted among 45- to 85-year-old patients in the Department of Stomatology and Internal Medicine of Shenzhen Center for Chronic Disease Control, including a healthy control group of 22 people, a systemically healthy control group of 19 people with periodontitis, a T2DM periodontal health group of 15 people, and a T2DM group of 21 people with periodontitis. The Florida periodontal probe was used for periodontal examination, and the clinical indexes, including probing depth (PD), clinical attachment level (CAL) and probing on bleeding (BOP), were recorded. The concentrations of HBD-2 and Ll-37 in gingival crevicular fluid were determined by ELISA. The differences in HBD-2, LL-37 and periodontal clinical indexes between the groups were compared, and correlation analysis was conducted.@*Results@#The PD values in T2DM with the periodontitis group were higher than those of the systemically healthy controls with periodontitis group (P < 0.05); the levels of HBD-2 and LL-37 in gingival crevicular fluid in systemically healthy controls with periodontitis group were significantly higher than those in the healthy control group (P < 0.05), the level of HBD-2 in gingival crevicular fluid in systemically healthy controls with periodontitis group was significantly higher than that in T2DM with periodontitis group (P < 0.05); and the antimicrobial peptides HBD-2 and LL-37 in gingival crevicular fluid were significantly positively correlated with the PD and CAL in systemically healthy controls with periodontitis group (P < 0.05), and there was no significant correlation between the antimicrobial peptides HBD-2, LL-37 in gingival crevicular fluid and PD, CAL in T2DM with periodontitis group (P > 0.05).@*Conclusion@#The levels of antimicrobial peptides HBD-2 and LL-37 in gingival crevicular fluid of middle-aged and elderly patients with T2DM periodontitis were lower, and there was no significant correlation with PD and CAL in periodontal clinical indicators.

Inhibitory effect of antimicrobial peptide LL-37 on tumor growth of mice with colon cancer and its mechanism / 吉林大学学报(医学版)

Objective: To investigate the effects of antimicrobial peptide LL-37 on the tumor growth and apoptosis of the mice with colon cancer, and to elucidate the possible molecular mechanism of its anti-tumor effect. Methods: The LL-37 over-expression colon cancer HT-29 cells were constructed, and the expression levels of LL-37 mRNA and protein in the HT-29 cells were detected by qRT-PCR and Western blotting methods. A total of 30 BALB/c mice were randomly divided into control group (given the uninfected HT-29 cells)' empty vector group (given the HT-29 cells infected with empty plasmid), LL-37 over-expression group (given the HT-29 cells infected with LL-37 over-expression vector), AMPK inhibitor group [given the HT-29 cells infected with empty vector, and then injected with 2 mg • kg Dorsomorphin (Dor) in the tail vein

Anti-tumor effect of new LL-37 hybrid antimicrobial peptide on breast cancer MCF-7 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective:To investigate the anti-tumor effect and mechanism of new LL-37 hybrid peptide on breast cancer MCF-7 cells. Methods: Human antimicrobial peptide LL-37 and human neutrophil peptide 1(HNP-1) were screened by using of Antimicrobial Peptides Database (http:// aps.unmc.edu/AP/main.php). The new LL-37 hybrid peptide was synthesized by integrating the active fragments, which were selected by bioinformatics analysis. The breast cancer MCF-7 cells and human normal breast MCF10A cells were treated with the new LL-37 hybrid peptides (0~70 μmol/L). Cell viability was monitored by CCK-8 assay and the affinity of the new LL-37 hybrid peptide with MCF-7 cells was observed using confocal laser scanning microscope (CLSM). The effects of LL-37 and caspase inhibitor on apoptosis and cell cycle of MCF-7 cells were measured by FCM (flow cytometry). Results: The new LL-37 hybrid peptide, as an amphiphilic cationic polypeptide, could selectively inhibit the proliferation of breast cancer MCF-7 cells ( P <0.05) with an IC50of 58.34 μmol/L, but exerted no significant effect on normal breast MCF10A cells. LL-37 peptide had high affinity with MCF-7 cells, which could cause S-stage stagnation and significantly increased early apoptosis ( P <0.01); however, the cell cycle block and apoptosis were significantly attenuated after the treatment of caspase inhibitor ( P <0.01). Conclusion: The new LL-37 hybrid peptide has anti-tumor activity on breast cancer MCF-7 cells, and could induce MCF-7 cells apoptosis possibly by arresting cell cycle via the caspase-dependent signaling pathway.

Serum level of cathelicidin LL-37 is increased in euthymic patients with bipolar disorder irrespective of their cardio-metabolic status

Abstract Background Antimicrobial peptides are components of the innate immune system. Cathelicidin LL-37 plays an important role in antimicrobial defense, exerts proinflammatory effect and strongly affects the immune system functioning. Our recent study revealed that serum concentration of LL-37 is increased in patients with bipolar disorder. Objectives The aim of this study is to re-evaluate serum LL-37 levels in patients with euthymic bipolar disorder and in healthy controls, matched for anthropometric and body composition parameters. Methods 36 adult patients with euthymic bipolar disorder and 68 non-depressed adults were included into the study. Concentration of LL-37 in serum was assessed using ELISA method. Detailed anthropometric measurements, body composition and biochemical analyses were performed. Results There was a statistically significant difference (p = 0.01) in serum LL-37 level between patients with bipolar disorder (4.97 ± 7.98 ng/mL) and control subjects (1.78 ± 2.69 ng/mL). Discussion Results of this study indicate that LL-37 serum level is increased in euthymic bipolar disorder patients. We found that this increase could not be attributed to analyzed anthropometric or body composition parameters.

Association of vitamin D3, VDR gene polymorphisms, and LL-37 with a clinical form of Chagas Disease

Abstract INTRODUCTION: Chagas disease (CD) is an important public health problem in Brazil and worldwide. Aging and obesity are important matters in patients with CD, as is hypovitaminosis D3, which can decrease the quality of life of these patients. Immunomodulation mediated by vitamin D3, especially the production of antimicrobial peptides such as cathelicidin LL-37, might be related to the severity and symptoms of CD. This study aimed to determine the serum levels of vitamin D and LL-37 and VDR gene polymorphisms in patients with chronic CD. METHODS: This study included male patients with cardiac and indeterminate clinical forms of CD. Clinical, anthropometric, and blood parameters were obtained. Serum levels of 25(OH)D3 and LL-37 were determined by chemiluminescence and enzyme-linked immunosorbent assay respectively. Fok (rs731236), Bsm (rs1544410), Apa (rs7975232), and Taq (rs731236) polymorphisms of the VDR gene were investigated by PCR-RFLP. RESULTS: Sixty-four patients were included in the study: 18 of the cardiac form and 46 of the indeterminate form. No differences in age, ethnicity, BMI, arterial hypertension, diabetes mellitus, or dyslipidemias were observed between groups. However, the serum levels of 25(OH)D3, but not of LL-37, were lower in the cardiac form group. The association among polymorphisms, vitamin D, and clinical form was not significant. CONCLUSIONS: Decreased levels of vitamin D suggest an association with the cardiac form of CD. Studies investigating the roles of vitamin D and LL-37 in the immune response and their associations with VDR polymorphisms and disease susceptibility are necessary.

Clinical significance of antibacterial peptide LL-37 in early diagnosis of patients with sepsis in emergency department / 中华危重病急救医学

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

Clinical significance of antibacterial peptide LL-37 in early diagnosis of patients with sepsis in emergency department / 中华危重病急救医学

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

Changes and clinical significance of antimicrobial peptide LL-37 and CRP in patients with Pseudomonas aeruginosa positive sputum culture / 国际检验医学杂志

Objective To investigate the changes of antimicrobial peptide LL-37 and C-reactive protein (CRP ) in the patients with Pseudomonas aeruginosa positive sputum culture and their mutual relation . Methods Fifty cases of Pseudomonas aeruginosa positive sputum culture and 27 cases undergoing physical ex-amination in the Guangdong Provincial Hospital of Chinese Medicine from September 2016 to May 2017 were selected as the research subjects .The level of antimicrobial peptide LL-37 was detected by double antibody sandwich ELISA and the CRP level was detected by immunoturbidimetry .Results The levels of antimicrobial peptide LL-37 and CRP in the Pseudomonas aeruginosa positive sputum culture group were significantly high-er than those in the healthy control group ,the difference was statistically significant (P< 0 .05) ,and they showed the positive correlation (r=0 .411 ,P<0 .05) .Conclusion In positive sputum culture of Pseudomonas aeruginosa ,antimicrobial peptide LL-37 and CRP levels are increased ,which has a certain clinical application value for the early diagnosis of infection .

Evaluation value of human antibacterial peptide LL-37 on the prognosis of elderly patients with sepsis / 中华危重病急救医学

Objective To evaluate the prognostic value of human antibacterial peptide LL-37 in elderly patients with sepsis. Methods Elderly sepsis patients over 65-year-old satisfied the diagnostic criteria for sepsis and septic shock admitted to intensive care unit of East Hospital of Tongji University from January 2016 to December 2017 were enrolled (elderly sepsis group). Aged community-acquired pneumonia (CAP) patients hospitalized during the same period were enrolled as a control group for pneumonia, and the aged health check-ups served as a healthy control group during the same period. The peripheral blood LL-37 levels of all patients on the 1st, 3rd, 7th day of admission and the results on the day of physical examination in the healthy control group and on the day of admission in aged CAP group were recorded. C-reactive protein (CRP), arterial blood lactate (Lac), procalcitonin (PCT) were monitored, and acute physiology and chronic health evaluation Ⅱ (APACHEⅡ) and sequential organ failure assessment (SOFA) scores were calculated based on the worst values within 24 hours. The correlation between LL-37 and various indicators was analyzed by Spearman method. According to the 28-day clinical outcome, the elderly patients with sepsis were divided into survival group and non-survival group. The differences in all parameters between the two groups were compared. The statistically significant indicators were analyzed by receiver operating characteristic (ROC) curve, and the predictive value of each indicator for prognosis was evaluated. Results ① A total of 113 elderly patients with sepsis were enrolled in the final analysis, including 67 patients in sepsis group and 46 patients in septic shock group. Thirty-two patients were enrolled as healthy controls and 31 elderly patients with CAP as elderly pneumonia group. The PCT, CRP, Lac, APACHEⅡ and SOFA scores of the patients in the three groups were higher than those of the healthy control group, and they were gradually increased with the severity of infection. There was no significant difference in gender or age among the groups. Compared with the healthy control group, the other three groups had higher LL-37 level after admission, the LL-37 levels in the sepsis group and the septic shock group were decreased with the prolongation of the hospitalization time, and they were lower than the pneumonia group at 7 days after admission [LL-37 (μg/L): 1 403.9±501.9, 1 517.1±676.4 vs. 1 608.4±816.2, both P > 0.05]. It was shown by correlation analysis that the LL-37 level in peripheral blood of elderly patients with sepsis was significantly negatively correlated with APACHEⅡ score (r = -0.329, P = 0.007) and SOFA score (r = -0.344, P = 0.005), but no significant correlation with Lac was found (r = -0.128, P = 0.311). ② The 28-day survival analysis revealed that of the 113 elderly patients with sepsis, 54 (47.8%) survived at 28 days and 59 (52.2%) died. There was no significant difference in gender, age, PCT or CRP levels at 1 day after admission between the two groups. The 1-day Lac, APACHEⅡ and SOFA scores of the patients in the non-survival group were significantly higher than those in the survival group, they were gradually increased with the prolongation of the hospitalization time, and they were significantly higher than those in the survival group at 7 days after admission [Lac (mmol/L): 2.4 (1.4, 4.4) vs. 1.0 (0.8, 1.7), APACHEⅡ score: 21.77±5.85 vs. 13.74±4.99, SOFA score: 9.62±4.78 vs. 3.18±2.71, all P < 0.01]. With the prolongation of admission, there was no significant change in LL-37 level of peripheral blood in the survival group. The LL-37 level in the non-survival group showed a downward tendency, and it was significantly lower than that in the survival group at 7 days after admission (μg/L: 1 277.8±642.6 vs. 1 620.6±461.6, P < 0.05). It was shown by ROC curve analysis that the LL-37 in peripheral blood, Lac, APACHEⅡ score and SOFA score at 7-day of admission of elderly patients with sepsis had predictive value for prognosis, and LL-37 had the best predicted effect for 28-day death, the area under the ROC curve (AUC) of LL-37 was 0.670, 95% confidence interval (95%CI) = 0.513-0.757, when the optimal cut-off value was 1 283.0 μg/L, the sensitivity was 75.7%, and the specificity was 61.5%. Conclusions The expression of LL-37 increased in the early course of the disease in elderly patients with sepsis. However, as the disease progressed and worsened, the level of LL-37 had a decline tendency and was associated with death. The dynamic monitoring of LL-37 combined with APACHEⅡ and SOFA scores had clinical guidance value in predicting the prognosis of sepsis in the elderly.

Antimicrobial peptide LL-37 in macrophages promotes colorectal cancer growth / 中华肿瘤杂志

Objective@#To investigate the effect and molecular mechanism of antimicrobial peptide LL-37 secreted by stromal cells on the growth of colorectal cancer cells.@*Methods@#Colorectal cancer cells SW480 or HCT116 were co-cultured with human macrophages using Transwell® maxicell inserts to mimic the tumor microenvironment. The effect of macrophages on the proliferation of colorectal cancer cells was detected by Bromodeoxyuridine and enzyme-linked immunosorbent assay (BrdU-ELISA). The expression of LL-37 mRNA and protein in macrophages and colorectal cancer cells was evaluated by reverse transcription-real-time quantitative PCR (RT-qPCR) and Western blot. LL-37 neutralizing antibody was added to abrogate the LL-37 activation. Additionally, macrophages were transfected with LL-37 shRNA plasmids to inhibit LL-37 expression. And then, the proliferation of colorectal cancer cells was observed. Furthermore, the growth-related signaling pathways were detected by Western blot.@*Results@#The BrdU-ELISA results showed that the absorbance of SW480 cells increased from 1.072±0.097 to 5.121±0.407 after co-culture (P<0.001), and that of HCT116 cells increased from 1.229±0.073 to 3.495±0.228 (P<0.001). RT-qPCR results showed that LL-37 mRNA expression in macrophages significantly increased from 2.682±0.191 to 6.117±0.768 after co-incubation (P<0.05), whereas that in SW480 had no significant difference. Consistently the protein expression of LL-37 in macrophages was significantly increased by Western blot, while it did not change in SW480. The proliferation rate of SW480 cells was repressed by adding LL-37 neutralizing antibody or LL-37 shRNA plasmid. Furthermore, Western blot analysis showed that the expression of non-phosphorylated (activated) β-catenin and its target genes cyclin D1 as well as c-myc were distinctly increased in co-cultured SW480 cells, which could be reversed by anti-LL-37 antibodies.@*Conclusion@#Macrophages promote the in vitro proliferation of colorectal cancer cells by enhancing the expression and secretion of antimicrobial peptides LL-37, and it seems that LL-37 activates colorectal cancer cells via Wnt/β-catenin pathway.

The role of LL-37 in tumor development / 中国肿瘤生物治疗杂志

@# LL-37是人体内发现的唯一一种Cathelicidin类抗菌肽，由其前体hCAP-18经丝氨酸蛋白酶3剪切后产生。研究发现， LL-37可在卵巢癌、肺癌、恶性黑色素瘤、皮肤鳞状细胞癌、前列腺癌等肿瘤中发挥促癌作用；而在胃癌、结直肠癌、白血病等肿瘤 中发挥抑癌作用。本文就LL-37在这些恶性肿瘤发生发展过程中所起的作用作一综述。

Effects of artesunate on rosacea-like inflammation in mouse models / 中华皮肤科杂志

Objective To evaluate effects of artesunate on rosacea-like inflammation in mouse models.Methods Twenty-five male BALB/c mice aged 7 weeks were injected subcutaneously with 40 μ1 antibacterial peptide LL-37 into the back once every 12 hours for 4 sessions to establish mouse models with rosacea-like inflammation.These 25 mice were randomly and equally divided into 5 groups:after each injection of LL-37,model group were gavaged with sodium chloride physiological solution,treatment groups gavaged with 25,50 and 100 mg/kg artesunate solution separately,and positive control group gavaged with 30 mg/kg doxycycline hydrochloride solution.Another 5 healthy mice injected subcutaneously with pure water into the back for 4 sessions served as blank control group.Forty-eight hours after the initial injection of LL-37,changes in skin lesions and the intensity of erythema were assessed.Skin tissues at the dorsal injection site were resected and subjected to HE staining,the tissue structure was observed and the number of inflammatory cells was counted.Enzyme-linked immunosorbent assay (ELISA) was performed to estimate the activity of myeloperoxidase (MPO) in skin lesions.Results The model group showed obvious inflammatory reactions,and significantly increased erythema score (3.20 ± 0.84),inflammatory cell count (517.27 ± 99.43) and MPO activity (0.57 ± 0.08) compared with the blank control group (all P ＜ 0.01).The positive control group showed significantly decreased erythema score (1.60 ± 0.89),inflammatory cell count (270.93 ± 124.63) and MPO activity (0.40 ± 0.05) compared with the model group (P ＜ 0.05,0.01,0.01,respectively).Moreover,the erythema score,inflammatory cell counts and MPO activity were all significantly lower in 50-(1.80 ± 0.84,286.00 ± 33.72,0.43 ± 0.05,respectively) and 100-mg/kg artesunate groups (1.40 ± 0.55,258.00 ± 36.44,0.40 ± 0.06,respectively) than in the model group (P ＜ 0.05 or 0.01).However,there were no significant differences in the erythema score,inflammatory cell count and MPO activity between 50-or 100-mg/kg artesunate group and the positive control group (P ＞ 0.05).Conclusion Artesunate can inhibit rosacea-like inflammatory reactions in mouse models,especially the middle-and high-dose artesunate.

LL-37 inhibited secretion of inflammatory cytokines in mycobacterium tuber-culosis infected macrophages / 中国免疫学杂志

Objective:To research the anti-inflammatory effect of LL-37 in mycobacterium tuberculosis ( Mtb ) infected-macrophages,as well as its influence on the secretion of inflammatory cytokines. Methods:( 1 ) THP-1 cells were cultured and incubated with phorbol 12-myristate 13-acetate (PMA) to transform into an adherent macrophage-like state (macrophage,Mφ). Then the THP-1 cell derived macrophages were infected with mycobacterium tuberculosis,and then stimulated with different concentrations of LL-37. (2)The experiment was divided into following groups: ① Control group:THP-1+normal saline (NS);② Mtb group:THP-1+Mtb;③Mtb+LL-37 5 μg/ml group:THP-1+Mtb+5 μg/ml LL-37;④Mtb+LL-37 10 μg/ml group:THP-1+Mtb+10 μg/ml LL-37;⑤Mtb+LL-37 20 μg/ml group:THP-1+Mtb+20 μg/ml LL-37. (3) The mRNA expression levels of IL-12p40,TNF-α,IL-4,and IL-10 will be determined by Real-time PCR respectively at 6,12,24 and 48 hours. The secreted levels of IL-12p40,TNF-α,IL-4,and IL-10 will be determined by ELISA analysis respectively at 6,12,24 and 48 hours. Results:The mRNA expression levels and secreted levels of IL-12p40,TNF-α,IL-4 and IL-10 were increased in Mtb group than those in control group. The mRNA expression levels and secreted levels of pro-inflammatory cytokines IL-12p40 and TNF-α were decreased in the LL-37 groups than those in Mtb group. However,anti-inflammatory cytokines IL-4 and IL-10 mRNA expression levels and secreted levels were increased in the LL-37 groups than those in Mtb group. Conclusion:Exogenous LL-37 inhibited the secretion of inflammatory cytokines in macrophages during mycobacterium tu-berculosis infection. The effect is related to the concentrations of LL-37 and the stimulated time of macrophages which were infected with mycobacterium tuberculosis. The results will provide new insights into the treatment of Mtb infection.

Avaliação do peptídeo LL-37 em contato com células-tronco da polpa dentária / Evaluation of LL-37 Peptide in contact with stem cells from dental pulp

O peptídeo LL-37 (catelicidina derivada de humano), é liberado por algumas células humanas e capaz de neutralizar os tecidos com lipopolissacarídeo (LPS), além de atrair células da polpa, e induzir a angiogênese, características que o tornam um possível adjunto para a regeneração do complexo dentino-pulpar. O objetivo desse trabalho foi avaliar in vitro a biocompatibilidade do peptídeo LL-37 nas concentrações de 5 e 10 µg/mL, e sua possível atuação na diferenciação de células-tronco da polpa dentária (DPSC) para odontoblastoslike. Com esse propósito, foram avaliados: (a) a citotoxicidade, pelo teste MTT; (b) a genotoxicidade, através do ensaio do micronúcleo; (c) a produção e quantificação de óxido nítrico; (d) as fases do ciclo celular, por citometria; (e) a expressão de alguns genes associados à formação de tecido mineralizado, através do teste qRT-PCR; (f) o conteúdo de proteína total; (g) a atividade de fosfatase alcalina (ALP); e (h) a produção de sialofosfoproteína dentinária (DSPP), pelo ensaio imunoenzimático ELISA. Foi observado que as concentrações de 5 e 10 µg/mL de LL-37 não foram citotóxicas e ainda aumentaram, em geral, a viabilidade celular (p<0,05), sendo que os maiores valores de absorbância foram observados no 3° dia de contato. As concentrações testadas também não induziram genotoxicidade, após 7 dias de contato, tendo sido genotóxico apenas o grupo controle positivo (EMS) (p<0,05). Ainda, não foi observado diferença estatisticamente significativa na produção de nitrito, pelas células expostas ao LL-37 após 7 dias, em ambas as concentrações. A análise do ciclo celular, evidenciou maior porcentual de células na fase G0/G1, em todos os grupos (p<0,05). Quando estes foram comparados, foi observado maior quantidade de células na fase G0/G1 na concentração de 10 µg/mL de LL- 37 comparada ao grupo controle (p<0,05). Por outro lado, o grupo controle exibiu mais células na fase G2 e em mitose (M) que os grupos tratados com 5 e 10 µg/mL de LL-37 (p<0,05), e mais células na interfase (S) que o grupo tratado com 10 µg/mL de LL-37 (p<0,05). A análise da expressão gênica demonstrou que não houve aumento de expressão dos genes fosfatase alcalina, osteocalcina, osteopontina e Runx2 após tratamento com ambas as concentrações do peptídeo, no 3° dia. Além disso, não foi observado diferença estatisticamente significativa na ALP nos grupos tratados e controle, após 3 e 14 dias, enquanto o conteúdo de proteína total foi maior aos 14 dias nos grupos tratados com LL-37 (p<0,05) Ainda, aos 3 dias, a produção da proteína DSPP foi maior no grupo tratado com 10 µg/mL de LL-37 (p<0,05). Com base nesses resultados, pode-se concluir que o LL-37 é biocompatível nas concentrações testadas nesse trabalho, e ainda aumenta o número de células viáveis, principalmente em período inicial. Além disso, aos 3 dias, na concentração de 10 µg/mL, ele retarda o ciclo celular e aumenta a expressão da proteína DSPP, além de aumentar a síntese proteica aos 14 dias, o que indica que esse peptídeo pode desempenhar algum tipo de função na diferenciação odontoblástica (AU)

The LL-37 peptide (human derived cathelicidin) is released by some human cells and able of neutralizing the tissues that present lipopolysaccharide (LPS), as well as, attracts pulp cells and induces angiogenesis; characteristics that makes it a possible adjunct for regeneration of the dentin-pulp complex. The aim of this study was evaluate in vitro the biocompatibility of LL-37 in the concentrations of 5 and 10 µg/mL, and its possible performance in the differentiation of dental pulp stem cells (DPSC) into odontoblasts-like cells. For this purpose, it was evaluated: (a) the cytotoxicity by MTT assay; (b) the genotoxicity by the micronucleus test; (c) the production and quantification of nitric oxide; (d) the cell cycle, by flow cytometry; (e) the expression of genes associated with the mineralization by qRT-PCR; (f) the total protein content; (g) the alkaline phosphatase activity (ALP); and (h) the production of dentine sialofosfoprotein (DSPP) by indirect enzyme-linked immunosorbent assay (ELISA). It was observed that the concentrations of 5 and 10 µg/ml of LL-37 were not cytotoxic, in addition to they increased, in general, the cell viability (p<0,05). Moreover, higher absorbance values were observed on 3rd day of contact. After 7 days, the tested concentrations also did not induce genotoxicity, (p<0,05); only the positive control group (EMS) was genotoxic (p<0.05). Furthermore, there was not statistical significance in the nitrite production by the cells exposed to LL-37 for 7 days, in both concentrations. The cell cycle test showed higher percentage of cells in the phase G0/G1 in all groups (p<0.05). When they were compared, it was noticied that concentration of 10 ug/ml of LL-37 arrested the cells in G0/G1 compared to the control group (p<0.05). On the other hand, the control group, exhibited higher amount of cells in G2 and mitosis (M) than the others (p<0.05) and also higher number of cells in interfase (S) than the group treated with 10 µg/mL of LL-37 (p<0.05). On the 3rd day, the analysis of gene expression demonstrated no increase in the expression of the genes alkaline phosphatase, osteocalcin, osteopontin and Runx2, after treatment with both peptide concentrations. Furthermore, it was not observed statistical significance in the ALP in the treated and control groups after 3 and 14 days, while total protein content was higher in the groups treated with LL-37, at 14 days (p<0.05). On the 3rd day, the production of DSPP protein was higher in the group treated with 10 µg/mL of LL-37 (p<0.05). Based on these results, it can be concluded that LL-37 is biocompatible at these concentrations and increases the number of viable cells, especially in the initial period. Moreover, on the 3rd day, the concentration of 10 µg/mL arrests the cell cycle, and increases the expression of DSPP protein, in addition to raising the protein content at 14 days, which indicates that this peptide may present some kind of function in the odontoblastic differentiation(AU)

Vitamin D Status and Its Association with the SCORAD Score and Serum LL-37 Level in Korean Adults and Children with Atopic Dermatitis

BACKGROUND: Vitamin D insufficiency could be associated with the prevalence of atopic dermatitis (AD). OBJECTIVE: To examine vitamin D status and the relations between serum 25-hydroxyvitamin D levels, SCORAD score, serum LL-37 level, and body mass index (BMI) in Korean AD patients, and to explore whether these associations differ between adults and children. METHODS: Serum 25-hydroxyvitamin D levels, serum LL-37, and clinical features were analyzed in a total of 72 Korean patients with AD (39 adults and 33 children) and 140 healthy control subjects (70 adults and 70 children). RESULTS: Serum 25-hydroxyvitamin D levels were significantly reduced in children with AD (15.06+/-4.64 ng/ml) compared with normal children in the control group (16.25+/-6.60 ng/ml) (p=0.036). Significant inverse correlations were found between BMI and 25-hydroxyvitamin D level (r=-0.315, p=0.007) and between the SCORAD score and serum LL-37 level (r=-0.3, p=0.011) in the total AD patients. CONCLUSION: The results showed that serum vitamin D levels were lower in children with AD than in healthy children; however, the same relation was not observed between adults with AD and healthy adults. Serum 25-hydroxyvitamin D concentration was not significantly correlated with AD severity or serum LL-37 levels in our study population.

Increased Expression of Cathelicidin by Direct Activation of Protease-Activated Receptor 2: Possible Implications on the Pathogenesis of Rosacea

PURPOSE: Recent findings of increased cathelicidin protein and its proteolytic fragments in rosacea suggest a pathogenic role for cathelicidin in this disease. The relationship between cathelicidin and protease-activated receptor 2 (PAR-2) is therefore of interest, as PAR-2, expressed principally in keratinocytes, regulates pro-inflammatory cytokine expression in the skin. The purpose of this study was to determine the relationship between expression of PAR-2 and cathelicidin in rosacea and to test the effect of direct PAR-2 activation on cathelicidin expression in keratinocytes. MATERIALS AND METHODS: Samples from 40 patients with clinicopathologic diagnosis of rosacea and facial skin tissue samples from 20 patients with no specific findings or milium without inflammation were retrieved. Intensities of immunohistochemical staining for PAR-2 and cathelicidin were compared between normal and rosacea-affected skin tissues. Additionally, correlations between PAR-2 and cathelicidin staining intensities within rosacea patients were analyzed. In cultured keratinocytes, changes in PAR-2, cathelicidin, and vascular endothelial growth factor (VEGF) mRNA and protein were analyzed after treatment with PAR-2 activating peptide (AP). RESULTS: Cathelicidin expression was significantly higher in rosacea skin tissues than in normal tissues (p<0.001), while PAR-2 expression was not significantly higher in rosacea tissues than in normal skin tissues. A positive correlation between PAR-2 and cathelicidin within rosacea samples was observed (R=0.330, p=0.037). After treatment of PAR-2 AP, both mRNA and protein levels for PAR-2, cathelicidin, and VEGF significantly increased in cultured keratinocytes, compared with PAR-2 control peptide treatment. CONCLUSION: PAR-2 may participate in the pathogenesis of rosacea through activation of cathelicidin LL-37, a mediator of innate immune responses in the skin.

Measurement of vitamin D and cathelicidin (LL-37) levels in patients of psoriasis with co-morbidities.

Background: During the last decade, a lot of co-morbidities (diabetes, obesity, heart disease, etc.) have been described to be associated with psoriasis, but the exact link at the molecular level is not well-known. Researchers have shown molecular level changes in vitamin D pathway and its relationship to cathelicidin. Aims: To estimate the levels of cathelicidin (LL-37), and vitamin D in psoriasis patients with co-morbidities, and compare them with matched healthy controls. Methods: One hundred consecutive patients with stable plaque psoriasis (psoriasis area and severity index ≥10) with no systemic treatment in the past 3 months were investigated for the serum levels of vitamin D and LL-37, and compared with equal number of matched healthy volunteers. Results: The serum vitamin D levels were significantly lower in patients. Furthermore, the levels of serum LL-37were significantly high. Conclusion: Our study showed that the low serum levels of vitamin D, and higher blood levels of cathelicidin could form a molecular level clue in the pathogenesis of psoriasis patients, who are more likely to develop co-morbidities.

Anti-adenoviral Effects of Human Cationic Antimicrobial Protein-18/LL-37, an Antimicrobial Peptide, by Quantitative Polymerase Chain Reaction

PURPOSE: Antimicrobial peptides have an important role in self-protection of the ocular surface. Human cationic antimicrobial protein (hCAP)-18 is a linear, alpha-helical peptide that consists of a conserved pro-sequence called a cathelin-like domain and a C-terminal peptide named LL-37. We investigated the in vitro anti-adenoviral activity of hCAP-18/LL-37 in several adenovirus types, inducing keratoconjunctivitis. METHODS: A549 cells were used for viral cell culture, and human adenovirus (HAdV) types 3 (HAdV3, species B), 4 (species E), 8, 19a, and 37 (species D) were used. The cytotoxicity of LL-37 was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay to obtain 50% cytotoxic concentration. After pretreatment of A549 cells with serial dilutions of LL-37 for 24 hours, adenovirus was cultured for seven days, and adenoviral DNA was quantitatively measured by real-time polymerase chain reaction (PCR). RESULTS: The 50% effective concentration of LL-37 obtained by real-time PCR ranged between 118 and 270 microM. LL-37 showed a significant inhibitory effect on adenoviral proliferation in all adenovirus types except HAdV4 in a dose-dependent manner. CONCLUSIONS: LL-37 has significant inhibitory activity against HAdV3, 8, and 19, which induce keratoconjunctivitis. These results indicate that hCAP-18/LL-37 may be a possible candidate for the treatment of HAdV keratoconjunctivitis.