Lactoferrin and Human Neutrophil Protein (HNP) 1-3 Levels During the Neonatal Period in Preterm Infants.

Antimicrobial polypeptides (APPs) are part of the innate immune system, but their specific role in the context of preterm birth is not yet understood. The aim of this investigation was to determine the systemic expression of APPs, i.e., lactoferrin (LF) and human neutrophil protein (HNP) 1-3 in preterm infants in the period of highest vulnerability for infection and to correlate these biomarkers with short-term outcome. We therefore conducted a prospective two-center study including plasma samples of 278 preterm infants and 78 corresponding mothers. APP levels were analyzed on day 1, 3, 7, and 21 of life via enzyme-linked immunosorbent assay (ELISA). The levels of LF and HNP1-3 remained stable during the first 21 days of life and were not influenced by maternal levels. Elevated APP levels were found at day 1 in infants born to mothers with amniotic infection syndrome (AIS vs. no AIS, mean ± SD in ng/ml: LF 199.8 ± 300 vs. 124.1 ± 216.8, HNP 1-3 16,819 ± 36,124 vs. 8,701 ± 11,840; p = 0.021, n = 179). We found no elevated levels of APPs before the onset of sepsis episodes or in association with other short-term outcomes that are in part mediated by inflammation such as necrotizing enterocolitis (NEC) or retinopathy of prematurity (ROP). Interestingly, infants developing bronchopulmonary dysplasia (BPD) showed higher levels of HNP1-3 on day 21 than infants without BPD (13,473 ± 16,135 vs. 8,388 ± 15,938, n = 111, p = 0.008). In infants born without amniotic infection, levels of the measured APPs correlated with gestational age and birth weight. In our longitudinal study, systemic levels of LF and HNP 1-3 were not associated with postnatal infection and adverse short-term outcomes in preterm infants.

Diagnostic values of HNP 1-3 and procalcitonin levels in synovial fluid aspirates in the differential diagnosis between septic arthritis and noninfectious arthritis.

INTRODUCTION: Although early diagnosis of septic arthritis may reduce mortality rates, and limit unnecessary surgical interventions, clinical parameters alone are not adequate for making the diagnosis of septic arthritis. Therefore, relevant laboratory parameters are used to enhance diagnostic sensitivity. The aim of our study was to assist in making the diagnosis of septic arthritis, and prevent delays in the diagnosis. For this purpose; we aimed to determine the diagnostic values of human neutrophil peptides 1-3 (HNP 1-3) and procalcitonin (PCT) in synovial fluids of patients with arthritis. By comparing the HNP 1-3 and procalcitonin levels, as well as CRP, in synovial fluid aspirates, we evaluated the significance of these data in the differential diagnosis of septic arthritis from noninfectious arthritis. METHODS: A total of 67 adults consisting of 37 septic arthritis and 30 noninfectious arthritis patients were included in our study. As bioindicators; levels of HNP 1-3, PCT, synovial and serum CRP levels were found to have significant ROC areas in discriminating septic arthritis patients from noninfectious arthritis patients. RESULTS: As a result, synovial fluid HNP 1-3 levels were significantly higher in septic arthritis patients compared to noninfectious arthritis patients (p < 0.001). The sensitivity, specificity, and accuracy of HNP 1-3 levels in the diagnosis of septic and noninfectious arthritis were found as 86%, 87%, and 87%, respectively (AUC of the ROC curve = 0.828). CONCLUSIONS: It was decided that the level of HNP 1-3 in the synovial fluid can be used as an alternative indicator in the diagnosis of septic arthritis.

Kynurenic Acid Analog Attenuates the Production of Tumor Necrosis Factor-α, Calgranulins (S100A 8/9 and S100A 12), and the Secretion of HNP1-3 and Stimulates the Production of Tumor Necrosis Factor-Stimulated Gene-6 in Whole Blood Cultures of Patients With Rheumatoid Arthritis.

Objectives: Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease with complex pathogenesis involving a variety of immunological events. Recently, it has been suggested that kynurenic acid (KYNA) might be a potential regulator of inflammatory processes in arthritis. KYNA has a definitive anti-inflammatory and immunosuppressive function. The aim of the present study is to investigate the complex effects of a newly synthesized KYNA analog-SZR72 on the in vitro production of tumor necrosis factor-α (TNF-α), tumor necrosis factor-stimulated gene-6 (TSG-6), calprotectin (SA1008/9), SA100 12 (EN-RAGE), and HNP1-3 (defensin-α) in the peripheral blood of patients with RA and the various effects of the disease. Methods: Patients with RA (n = 93) were selected based on the DAS28 score, medication, and their rheumatoid factor (RF) status, respectively. Peripheral blood samples from 93 patients with RA and 50 controls were obtained, and activated by heat-inactivated S. aureus. Parallel samples were pretreated before the activation with the KYNA analog N-(2-N, N-dimethylaminoethyl)-4-oxo-1H-quinoline-2-carboxamide hydrochloride. Following the incubation period (18 h), the supernatants were tested for TNF-α, TSG-6, calprotectin, S100A12, and HNP1-3 content by ELISA. Results: SZR72 inhibited the production of the following inflammatory mediators: TNF-α, calprotectin, S100A12, and HNP1-3 in whole blood cultures. This effect was observed in each group of patients in various phases of the disease. The basic (control) levels of these mediators were higher in the blood of patients than in healthy donors. In contrast, lower TSG-6 levels were detected in patients with RA compared to healthy controls. In addition, the KYNA analog exerted a stimulatory effect on the TSG-6 production ex vivo in human whole blood cultures of patients with RA in various phases of the disease. Conclusion: These data further support the immunomodulatory role of KYNA in RA resulting in anti-inflammatory effects and draw the attention to the importance of the synthesis of the KYNA analog, which might have a future therapeutic potential.

Diminished Systemic and Mycobacterial Antigen Specific Anti-microbial Peptide Responses in Low Body Mass Index-Latent Tuberculosis Co-morbidity.

Low body mass index (BMI) is a risk factor for progression from latent Mycobacterium tuberculosis infection to active tuberculosis (TB) disease. Anti-microbial peptides (AMPs) are multifunctional molecules that play a crucial role in the mammalian host innate defense mechanism. AMPs have been shown to have an important role in host immunity to TB infection. The association of antimicrobial peptides with low BMI-latent tuberculosis (LTBI) co-morbidity has not been explored. To study the association of AMPs with LTBI-BMI, we examined the systemic, baseline, and mycobacterial antigen stimulated levels of human neutrophil peptides 1-3, (HNP1-3), granulysin, human beta defensin-2 (HBD-2), and cathelicidin (LL-37) in individuals with LTBI and low BMI (LBMI) and compared them with individuals with LTBI and normal BMI (NBMI). LBMI was characterized by diminished systemic levels of HNP1-3, granulysin, HBD-2 and cathelicidin in comparison with NBMI. Similarly, LBMI was also characterized by diminished unstimulated levels of HNP1-3 and granulysin and diminished mycobacterial antigen stimulated levels of HNP1-3, granulysin, and HBD-2. In addition, certain AMPs exhibited a positive correlation with BMI. Our data, therefore, demonstrates that coexistent LBMI in LTBI is characterized by the diminished levels of HNP1-3, granulysin, HBD-2, and cathelicidin, thereby potentially increasing the risk of progression to active TB.

Salivary Levels of Antimicrobial Peptides in Chronic Periodontitis Patients with Type 2 Diabetes.

OBJECTIVE: The aim of this study was to evaluate the interrelationship between salivary levels of human neutrophil antimicrobial peptides (AMP) 1-3 (HNP 1-3), LL-37 and periodontitis in individuals with and without type 2 diabetes (T2DM). METHODS: Eighty individuals were enrolled and grouped as follows: Group I: 20 healthy individuals, Group II: 20 systemically healthy individuals with chronic periodontitis (CP), Group III: 20 individuals with T2DM only and Group IV: 20 individuals T2DM and CP. Plaque index, probing pocket depth, bleeding on probing and clinical attachment levels were evaluated. The diabetic status was established by assessing the levels of fasting plasma glucose and HbA1c. Salivary levels of HNP 1-3 and LL-37 were assessed by ELISA. RESULTS: The present study demonstrated that individuals with T2DM and CP had significantly higher salivary levels of LL-37 (443.00 ±221.52 ng/ml) and HNP 1-3 (149.52 ± 35.07 ng/ml) (p less than 0.001) compared to other groups. Additionally, salivary LL-37 and HNP 1-3 were significantly correlated with clinical and laboratory parameters (p less than 0.001). A significant positive correlation was seen between salivary levels of LL-37 and HNP 1-3 (r=0.69; p less than 0.001). CONCLUSIONS: LL-37 concentrations were highest in patients with T2DM+CP when compared with controls. LL-37 was positively correlated with age. HNP 1-3 levels were increased in groups with DM when compared to the groups without periodontitis. The role of AMPs is vital in the immunoinflammatory response in the pathogenesis of periodontitis and DM.

Effects of Protein Versus Carbohydrate Supplementation on Markers of Immune Response in Master Triathletes: A Randomized Controlled Trial.

Objective: This study examines the long-term effects of ingesting hydrolyzed beef protein versus carbohydrate on indirect markers of immunity during 10 weeks of endurance training in master-aged triathletes (n = 16, age 35-60 years). Methods: Participants were randomly assigned to either a hydrolyzed beef protein (PRO, n = 8) or nonprotein isoenergetic carbohydrate (CHO, n = 8) condition, which consisted of ingesting 20 g of each supplement, mixed with water, once a day immediately post workout, or before breakfast on nontraining days. Salivary human neutrophil peptides (HNP1-3) were measured before and after performing an incremental endurance test to volitional exhaustion at both pre and post intervention. Additionally, baseline levels of platelets, neutrophils, eosinophil basophils, monocytes, and lymphocytes were determined at pre and post intervention. Results: No significant changes in baseline concentration and secretion rate of salivary HNP1-3 were observed for either treatment. The CHO group showed a nonsignificant decrease in resting HNP1-3 concentrations following the intervention (p = 0.052, effect size d = 0.53). Protein supplementation demonstrated a significant reduction in lymphocyte counts pre to post intervention (mean [SD]: 2.30 [0.57] vs. 1.93 [0.45] 103/mm3, p = 0.046, d = 0.77), along with a moderate but not statistically significant increase (d = 0.75, p = 0.051) of the neutrophil-to-lymphocyte ratio. Conclusions: In master-aged triathletes, postworkout ingestion of only protein, with no carbohydrate, may not be as effective as carbohydrate alone to attenuate negative long-term changes of some salivary and cellular immunological markers. Future studies should consider the co-ingestion of both macronutrients.

Increased alpha-defensin expression is associated with risk of coronary heart disease: a feasible predictive inflammatory biomarker of coronary heart disease in hyperlipidemia patients.

BACKGROUND: Atherosclerosis is a multifactorial disorder of the heart vessels that develops over decades, coupling inflammatory mechanisms and elevated total cholesterol levels under the influence of genetic and environmental factors. Without effective intervention, atherosclerosis consequently causes coronary heart disease (CHD), which leads to increased risk of sudden death. Polymorphonuclear neutrophils play a pivotal role in inflammation and atherogenesis. Human neutrophil peptides (HNPs) or alpha (α)-defensins are cysteine-rich cation polypeptides that are produced and released from activated polymorphonuclear neutrophil granules during septic inflammation and acute coronary vascular disorders. HNPs cause endothelial cell dysfunction during early atherogenesis. In this cross-sectional study, control, hyperlipidemia and CHD groups were representative as atherosclerosis development and CHD complications. We aimed to assess the correlation between α-defensin expression and the development of CHD, and whether it was a useful predictive indicator for CHD risk. METHODS: First, DNA microarray analysis was performed on peripheral blood mononuclear cells (PBMCs) from Thai control, hyperlipidemia and CHD male patients (n = 7). Gene expression profiling revealed eight up-regulated genes common between hyperlipidemia and CHD patients, but not controls. We sought to verify and compare α-defensin expression among the groups using: 1) real-time quantitative RT-PCR (qRT-PCR) to determine α-defensin mRNA expression (n = 10), and 2) enzyme-linked immunosorbent assay to determine plasma HNP 1-3 levels (n = 17). Statistically significant differences and correlations between groups were determined by the Mann-Whitney U test or the Kruskal-Wallis test, and the Rho-Spearman correlation, respectively. RESULTS: We found that α-defensin mRNA expression increased (mean 2-fold change) in the hyperlipidemia (p = 0.043) and CHD patients (p = 0.05) compared with the controls. CHD development moderately correlated with α-defensin mRNA expression (r = 0.429, p = 0.023) and with plasma HNP 1-3 levels (r = 0.486, p = 0.000). CONCLUSIONS: Increased α-defensin expression is a potential inflammatory marker that may predict the risk of CHD development in Thai hyperlipidemia patients.

The correlation between plasma human neutrophil peptide 1-3 levels and severity of coronary artery disease.

INTRODUCTION: Inflammation plays a key role in atherosclerosis, and discovering new biomarkers of inflammation is becoming important in order to uncover the pathogenesis of atherosclerotic coronary artery disease (CAD). Recent studies have focused on polymorphonuclear neutrophils. It has been suggested that human neutrophil peptide 1-3 (HNP1-3) is proatherogenic. In this study, we aimed to investigate the associations between plasma HNP1-3 levels and the severity of atherosclerosis via a generally accepted scoring system. MATERIAL AND METHODS: This cross-sectional, observational study included 107 consecutive patients suffering from stable angina pectoris and undergoing coronary angiography (CAG). Patients were divided into two groups according to the Gensini scoring (GS) system evaluating disease severity. Group 1 was composed of mild CAD patients with GS < 20 and group 2 consisted of severe CAD patients with GS ≥ 20. Plasma HNP1-3 levels were assessed by the ELISA method. RESULTS: The mean HNP1-3 levels were found to be lower in group 1 than group 2 (134.7 ng/ml vs. 147.5 ng/ml). HNP1-3 levels were significantly higher in the severe CAD group than the mild CAD group according to GS (p < 0.001). The results of multivariate logistic regression analysis revealed that only age > 62 years and HNP1-3 > 134 ng/ml were independent predictors of the severity of CAD after adjusting for gender, smoking, hypertension, hyperlipidemia, diabetes, family history of CAD and white blood cell count. In predicting the severity of CAD, the sensitivity and specificity of HNP1-3 were 83.9% (p < 0.001) and 58.8% (p < 0.001), respectively. CONCLUSIONS: This study revealed that the plasma levels of HNP1-3 were significantly higher in severe CAD than mild CAD.

Relevance of α-defensins (HNP1-3) and defensin ß-1 in diabetes.

AIM: To investigate the genetic background of human defensin expression in type 1 and 2 diabetes. METHODS: Associations between DEFA1/DEFA3 gene copy number polymorphism and diabetes as well as between the promoter polymorphisms of DEFB1 and diabetes were studied. The copy number variation of the DEFA1/DEFA3 genes was determined in 257 diabetic patients (117 patients with type 1 and 140 with type 2 diabetes). The control group consisted of 221 age- and gender-matched healthy blood donors. The cumulative copy numbers of the DEFA1/DEFA3 genes were detected by using quantitative PCR analysis. To evaluate the HNP 1-3 (human neutrophil peptide 1-3 or α-defensin) levels in the circulation, plasma HNP 1-3 concentrations were measured by ELISA. The expression of DEFA1/A3 in peripheral leukocytes of the diabetic patients was measured by quantitative RT PCR analysis. Three SNPs of the human DEFB1 (human defensin ß-1) gene: DEFB1 G-20A (rs11362), DEFB1 C-44G (rs1800972) and DEFB1 G-52A (rs1799946) were genotyped by Custom TaqMan(®) Real Time PCR assay. RESULTS: Significant differences were observed in HNP1-3 levels between the healthy subjects and both groups of diabetic patients. The mean ± SE was 28.78 ± 4.2 ng/mL in type 1 diabetes, and 29.82 ± 5.36 ng/mL in type 2 diabetes, vs 11.94 ± 2.96 ng/mL in controls; P < 0.01 respectively. There was no significant difference between patients with type 1 and type 2 diabetes in the high plasma concentrations of HNP1-3. The highest concentrations of α-defensin were found in diabetic patients with nephropathy (49.4 ± 4.8 ng/mL), neuropathy (38.7 ± 4.8 ng/mL) or cardiovascular complications (45.6 ± 1.45 ng/L). There was no significant difference in the cumulative copy numbers of DEFA1/DEFA3 genes between controls and patients, or between patients with the two types of diabetes. Comparisons of HNP 1-3 plasma level and DEFA1/A3 copy number of the same patient did not reveal significant relationship between defensin-α levels and the gene copy numbers (r (2) = 0.01). Similarly, no positive correlation was observed between the copy numbers and the mRNA expression levels of DEFA1/A3. Regarding the C-44G polymorphism of DEFB1, the GG "protective" genotype was much less frequent (1%-2%) among both groups of patients than among controls (9%). CONCLUSION: Elevated HNP1-3 levels in diabetes are independent of DEFA1/DEFA3 copy numbers, but GG genotype of C-44G SNP in DEFB1 gene may result in decreased defensin ß-1 production.