BACKGROUNDAntimicrobial peptides, including cathelicidin LL-37, human beta defensin (HBD)-2, and HBD-3, are important elements of the innate immune response and involved in modulation of the adaptive immunity, and they also play an important role in cutaneous defense against Mycobacterium tuberculosis.

METHODSThe fresh skin tissues and paraffin-embedded biopsy samples from three cutaneous tuberculosis, two tuberculids, and ten healthy individuals were collected. The expressions of LL-37, HBD-2, and HBD-3 mRNA in the lesions of three cutaneous tuberculosis and two tuberculids were detected by quantitative real-time polymerase chain reaction; the protein expressions were detected by immunohistochemistry and Western blotting methods.

RESULTSThe expressions of LL-37 mRNA and protein in the lesions of cutaneous tuberculosis and tuberculids were similar to that of normal skin. The expression of HBD-2 mRNA had an increasing trend in the lesions of cutaneous tuberculosis and tuberculids compared with that of normal skin; however, the expression of HBD-2 protein in the lesions of cutaneous tuberculosis had a decreasing trend compared with that of normal skin, and the expression of HBD-2 protein in the lesions of tuberculids was similar to that of normal skin. The expressions of HBD-3 mRNA and protein in lesions of cutaneous tuberculosis and tuberculids were similar to that of normal skin.

CONCLUSIONSOur study indicated that the expression of HBD-2 and HBD-3 mRNA and protein in lesions of cutaneous tuberculosis may be not consistent with that of tuberculids. However, an inherent limitation of the present study was that the sample size was small, and the roles and regulation mechanisms of LL-37, HBD-2, and HBD-3 in cutaneous tuberculosis and tuberculids need to be further investigated.

Adult ; Aged ; Antimicrobial Cationic Peptides ; genetics ; Female ; Humans ; Male ; Middle Aged ; RNA, Messenger ; analysis ; Tuberculosis, Cutaneous ; metabolism ; beta-Defensins ; genetics

OBJECTIVES: To study the value of heparin-binding protein (HBP) in the diagnosis of severe infection in children. METHODS: This study was a prospective observational study. The medical data of children who were admitted to the pediatric intensive care unit due to infection from January 2019 to January 2020 were collected. According to the diagnostic criteria for severe sepsis and sepsis, the children were divided into a severe sepsis group with 49 children, a sepsis group with 82 children, and a non-severe infection group with 33 children. The three groups were compared in terms of related biomarkers such as plasma HBP, serum C-reactive protein, serum procalcitonin, and platelet count. The receiver operating characteristic (ROC) curve was plotted to investigate the value of plasma HBP level in the diagnosis of severe infection (including severe sepsis and sepsis). RESULTS: The severe sepsis and sepsis groups had a significantly higher plasma HBP level on admission than the non-severe infection group (P<0.05). Compared with the sepsis and non-severe groups, the severe sepsis group had significantly higher serum levels of C-reactive protein and procalcitonin and a significantly lower platelet count (P<0.05). Plasma HBP level had an area under the ROC curve of 0.590 in determining severe infection, with a sensitivity of 38.0% and a specificity of 82.4% (P<0.05). CONCLUSIONS There is an increase in plasma HBP level in children with severe infection, and plasma HBP level has a lower sensitivity but a higher specificity in the diagnosis of severe infection and can thus be used as one of the markers for the judgment of severe infection in children.
Antimicrobial Cationic Peptides ; Biomarkers ; Blood Proteins ; C-Reactive Protein/analysis* ; Child ; Humans ; Procalcitonin ; Prospective Studies ; ROC Curve ; Sepsis/diagnosis*

OBJECTIVE: To explore the predictive value of heparin-binding protein (HBP) combined with sequential organ failure assessment (SOFA) score in patients with septic shock. METHODS: Seventy-eight patients with sepsis admitted to intensive care unit (ICU) of Henan Provincial People's Hospital from December 2016 to May 2017 were enrolled. Thirty healthy persons were enrolled as controls. The patient's gender, age, length of ICU stay, and blood culture results, white blood cell count (WBC), C-reactive protein (CRP), procalcitonin (PCT), blood lactate (Lac), HBP, SOFA score, acute physiology and chronic health evaluation II (APACHE II) score, organ failure and vasoactive agents usage within 24 hours of admission were recorded. The differences in the above indicators between the groups were compared, and the receiver operating characteristic (ROC) curve was drawn to evaluate the predictive value of HBP, SOFA score and their combination in patients with septic shock. RESULTS: All patients were enrolled in the final analysis, including 64 with sepsis and 14 with septic shock. Compared with the sepsis group, the proportion of patients with septic shock who were positive for blood culture, organ failure, and vasoactive agents was higher [57.1% (8/14) vs. 7.8% (5/64), 100.0% (14/14) vs. 65.6% (42/64), 100.0% (14/14) vs. 18.8% (12/64), all P < 0.01], SOFA and APACHE II scores were also higher (SOFA: 8.93±4.16 vs. 5.89±2.68, APACHE II: 22.29±4.89 vs. 15.28±5.14, both P < 0.01); however, there was no significant difference in gender, age or length of ICU stay between the two groups. Compared with the healthy control group, HBP, PCT, CRP and Lac levels were significantly increased in the sepsis group and the septic shock group. HBP in the septic shock group was significantly higher than that in the sepsis group (μg/L: 120.33±43.49 vs. 68.95±54.15, P < 0.05), but there was no significant difference in PCT, CRP or Lac between septic shock group and sepsis group [PCT (μg/L): 1.42 (0.47, 46.00) vs. 0.71 (0.19, 4.50), CRP (mg/L): 102.90±78.12 vs. 102.07±72.15, Lac (mmol/L): 1.81 (1.14, 3.65) vs. 1.59 (1.17, 2.24), all P > 0.05]. It was shown by ROC curve analysis that the area under the ROC curve (AUC) of SOFA score for predicting septic shock was 0.715 [95% confidence interval (95%CI) = 0.540-0.890, P = 0.012], and when the optimal cut-off value was 7.5, the sensitivity was 64.3%, the specificity was 76.6%. The AUC of HBP was 0.814 (95%CI = 0.714-0.913, P < 0.001), and when the optimal cut-off value was 89.43 μg/L, the sensitivity was 78.6%, the specificity was 76.6%; when the two were combined, the AUC was 0.829 (95%CI = 0.724-0.935, P < 0.001), the sensitivity was 92.9%, and the specificity was 61.9%. CONCLUSIONS HBP can be used as a biological indicator for predicting septic shock, and the accuracy of predicting septic shock can be improved with the combination of SOFA score.
Antimicrobial Cationic Peptides/analysis* ; Blood Proteins/analysis* ; Carrier Proteins/analysis* ; Female ; Humans ; Male ; Organ Dysfunction Scores ; Predictive Value of Tests ; Shock, Septic/diagnosis*

AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIR fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDESTl 5 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 F. coli transformed by the GST-AIR expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIR fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIR on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 F. coli with starting OD₆₀₀ at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.
Antimicrobial Cationic Peptides ; biosynthesis ; Antineoplastic Agents ; metabolism ; Escherichia coli ; metabolism ; Genetic Vectors ; HL-60 Cells ; Humans ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; Sequence Analysis, DNA

To screen the candidate genes associated with Musca domestica antibacterial peptides using DNA microarray technique, the hybrid probes were designed from the conservative domains of the encoded area of the insect antibacterial peptide genes in GenBank with biology software Designer 2.0, and were synthesized by a chemical process, with the assistance of the automated Gen III Microarray Spotter, those oligo probes were printed on a special ready-made glass, and a cDNA microarray was constructed. The total RNA was extracted from the fat body of Musca domestic third-instar larve induced after 24 hours by Escherichia coli and Staphylococcus aureus, the strands of cDNA were labled with fluoresceine Cy3 using the method of reverse transcription PCR, after prehybridization, hybridization and washing procedure, the results of hybridization were scanned using computer system, and the data were analyzed using the software of MIDAS, fifteen valid hybridization signals were detected through two times of hybridization and scanning (the positive samples as a control were excluded). DNA microarray technique can be successfully applied screen the candidate genes associated with Musca domestica antibacterial peptides, and further provide significant evidence to discover its antibacterial peptide new genes.
Animals ; Antimicrobial Cationic Peptides ; genetics ; Base Sequence ; Gene Expression Profiling ; Genes, Insect ; Houseflies ; genetics ; growth & development ; Larva ; genetics ; growth & development ; Molecular Sequence Data ; Oligonucleotide Array Sequence Analysis ; methods ; Oligonucleotide Probes ; chemistry

Defensins and cathelicidins (LL-37) are major antimicrobial peptides (AMPs) of the innate immune system of the human skin. In normal non-inflamed skin these peptides are negligible, but their expression can be markedly increased in inflammatory skin disease such as psoriasis. We designed this study to identify the expressions of LL-37 in normal human keratinocyte (NHK) and HaCaT cells after exposure to stimulants and to investigate difference of LL-37 expression accompanied with cell differentiation status, and come to understand difference of susceptibility to infection in atopic dermatitis and psoriasis. Expressions of LL-37 in NHKs and HaCaT cells were evaluated by using RT-PCR, Western blotting, and immunohistochemical (IHC) staining at 6, 12, and 24 hr post stimulation after exposure to Ultraviolet B irradiation and lipopolysaccharide. And expression of LL-37 in skin biopsy specimens from patients with atopic dermatitis and psoriasis was determined by immunohistochemical analysis. In time-sequential analyses of LL-37 expression revealed that LL-37 was expressed in NHKs, but not in HaCaT cells. IHC analysis confirmed the presence of abundant LL-37 in the epidermis of psoriasis. Therefore we deduced that expression of LL-37 is affected by UV irradiation, bacterial infection, and status of cell differentiation.
Antimicrobial Cationic Peptides/analysis/*genetics ; Blotting, Western ; Cell Line ; Cells, Cultured ; Comparative Study ; Defensins/analysis/genetics ; Dose-Response Relationship, Drug ; Gene Expression/drug effects/radiation effects ; Humans ; Immunohistochemistry ; Keratinocytes/cytology/*metabolism ; Lipopolysaccharides/pharmacology ; Male ; RNA, Messenger/genetics/metabolism ; Research Support, Non-U.S. Gov't ; Reverse Transcriptase Polymerase Chain Reaction ; Skin/cytology/metabolism ; Skin Diseases/*genetics/metabolism/pathology