Alpha-defensins Research Articles

Reversal of Azole Resistance in Candida albicans by Human Neutrophil Peptide.

With the spread of AIDS and the increase in immunocompromised patients, multi-drug-resistant fungal infections have become a serious concern among clinicians, predominantly in the developing world. Therefore, developing novel strategies and new drugs is essential to overcome drug resistance in fungal pathogens. Antimicrobial peptides of human origin have been investigated as a potential treatment against Candida infections. In this study, human neutrophil peptide (HNP) was tested for its antifungal activity alone and in combination with fluconazole (FLC) against azole-susceptible and resistant C. albicans isolates, following CLSI guidelines. Susceptibility and combination interactions were also confirmed by MUSE cell viability assay and isobolograms for synergistic combinations, respectively. The effect of HNP on biofilm inhibition was determined spectrophotometrically and microscopically. Drug susceptibility testing showed minimum inhibitory concentrations (MICs) and minimum fungicidal concentrations (MFCs) ranging from 7.813 to 62.5 µg/mL and 15.625 to 250 µg/mL against all the tested C. albicans strains. The combination activity of FLC with HNP exhibited synergistic and additive interactions in 43% of each and indifferent interaction in 14%, and none of the combinations showed antagonistic interaction. Furthermore, HNB inhibited biofilm formation in all the tested C. albicans isolates. At the respective MICs, HNP exhibited inhibitory effects on the activity of the drug efflux pumps and their genes. These results warrant the application of HNP as a mono- or combination therapy with FLC to treat azole-resistant C. albicans.

Induction of Endogenous Antimicrobial Peptides to Prevent or Treat Oral Infection and Inflammation

Antibiotics are often used to treat oral infections. Unfortunately, excessive antibiotic use can adversely alter oral microbiomes and promote the development of antibiotic-resistant microorganisms, which can be difficult to treat. An alternate approach could be to induce the local transcription and expression of endogenous oral antimicrobial peptides (AMPs). To assess the feasibility and benefits of this approach, we conducted literature searches to identify (i) the AMPs expressed in the oral cavity; (ii) the methods used to induce endogenous AMP expression; and (iii) the roles that expressed AMPs may have in regulating oral inflammation, immunity, healing, and pain. Search results identified human neutrophil peptides (HNP), human beta defensins (HBD), and cathelicidin AMP (CAMP) gene product LL-37 as prominent AMPs expressed by oral cells and tissues. HNP, HBD, and LL-37 expression can be induced by micronutrients (trace elements, elements, and vitamins), nutrients, macronutrients (mono-, di-, and polysaccharides, amino acids, pyropeptides, proteins, and fatty acids), proinflammatory agonists, thyroid hormones, and exposure to ultraviolet (UV) irradiation, red light, or near infrared radiation (NIR). Localized AMP expression can help reduce infection, inflammation, and pain and help oral tissues heal. The use of a specific inducer depends upon the overall objective. Inducing the expression of AMPs through beneficial foods would be suitable for long-term health protection. Additionally, the specialized metabolites or concentrated extracts that are utilized as dosage forms would maintain the oral and intestinal microbiome composition and control oral and intestinal infections. Inducing AMP expression using irradiation methodologies would be applicable to a specific oral treatment area in addition to controlling local infections while regulating inflammatory and healing processes.

LL-37 Triggers Antimicrobial Activity in Human Platelets.

Platelets play a crucial role in hemostasis and the immune response, mainly by recognizing signals associated with vascular damage. However, it has recently been discovered that the antimicrobial peptide LL-37 activates platelets in functions related to thrombus formation and inflammation. Therefore, this work aims to evaluate the effect of LL-37 on the activation of antimicrobial functions of human platelets. Our results show that platelets treated with LL-37 increase the surface expression of receptors (Toll-like receptors (TLRs) 2 and -4, CD32, CD206, Dectin-1, CD35, LOX-1, CD41, CD62P, and αIIbβ3 integrins) for the recognition of microorganisms, and molecules related to antigen presentation to T lymphocytes (CD80, CD86, and HLA-ABC) secrete the antimicrobial molecules: bactericidal/permeability-increasing protein (BPI), azurocidin, human neutrophil peptide (HNP) -1, and myeloperoxidase. They also translate azurocidin, and have enhanced binding to Escherichia coli, Staphylococcus aureus, and Candida albicans. Furthermore, the supernatant of LL-37-treated platelets can inhibit E. coli growth, or platelets can employ their LL-37 to inhibit microbial growth. In conclusion, these findings demonstrate that LL-37 participates in the antimicrobial function of human platelets.

P169 Copy number variation of alpha defensin 1-3 is a predictive marker of therapeutic response in ulcerative colitis

Abstract Background Alpha-defensin 1-3 (DEFA1A3) is an antimicrobial peptide found in phagocytic cells such as neutrophils, functioning as an effector factor in innate immunity. Reports suggest that copy number variation (CNV) of the DEFA1A3 gene is associated with the severity of ulcerative colitis (UC). However, the impact of DEFA1A3 on the pathogenesis of UC, and treatment response has not been fully investigated. Therefore, we aimed to evaluate the relationship between DEFA1A3-CNV and UC treatment response. Methods This was a retrospective and two medical centers study. The study included UC patients with DEFA1A3-CNV measurements, who attended our department and related facilities between 2020 and 2022. Clinical background and white blood cell count and serological marker were collected from the medical records of included patients. DEFA1A3-CNV was measured using droplet digital PCR after DNA extraction from peripheral blood mononuclear cells. Mayo score was used to determine the response to treatment and severity of UC. Results In total, 197 UC patients were included in the study. The median DEFA1A3 copy number was 7.36, and there was a significant correlation between DEFA1A3 copy number and Mayo score. The high DEFA1A3 copy number group had a significantly higher severity of UC during the observation period, compared with the low copy number group. In the analysis of the relationship between prednisolone/immunosuppressive therapy and DEFA1A3-CNV, the DEFA1A3 copy number was significantly higher in the non-remission group than in the remission group following prednisolone treatment. However, there was no correlation between other immunosuppressive therapies and DEFA1A3 copy number. Conclusion DEFA1A3 copy number is a predictive marker of disease severity and prednisolone treatment response.

Plasma human neutrophil peptides as biomarkers of disease severity and mortality in patients with decompensated cirrhosis.

Human neutrophil peptides (HNP)-1, -2 and -3 are the most abundant proteins in neutrophil azurophilic granules and are rapidly released via neutrophil degranulation upon activation. The aims of our study were to assess the role of HNP1-3 as biomarkers of disease severity in patients with decompensated cirrhosis and their value in predicting short-term mortality. In this study, 451 patients with acutely decompensated cirrhosis (AD) were enrolled at the two medical centres. Overall, 281 patients were enrolled as the training cohort from October 2015 to April 2019, and 170 patients were enrolled as the validation cohort from June 2020 to February 2021. Plasma HNP1-3 levels were measured using enzyme-linked immunosorbent assay (ELISA). Plasma HNP1-3 increased stepwise with disease severity (compensated cirrhosis: 0.3 (0.2-0.4); AD without acute-on-chronic liver failure (ACLF): 1.9 (1.3-4.8); ACLF-1: 2.3 (1.8-6.1); ACLF-2: 5.6 (2.9-12.3); ACLF-3: 10.3 (5.7-17.2)ng/ml). From the multivariate Cox regression analysis, HNP1-3 emerged as independent predictors of mortality at 30 and 90 days. Similar results were observed in the subgroup analysis. On ROC analysis, plasma HNP1-3 showed better predictive accuracy for 30- and 90-day mortality (area under the receiver operating characteristic (AUROC) of 0.850 and 0.885, respectively) than the neutrophil-to-lymphocyte ratio (NLR) and similar accuracy as end-stage liver disease (MELD: 0.881 and 0.874) and chronic liver failure-sequential organ failure (CLIF-SOFA: 0.887 and 0.878). Plasma HNP1-3 levels were closely associated with disease severity and might be used to identify patients with AD at high risk of short-term mortality.

Salivary Human β-Defensin 1-3 and Human α-Defensin-1 Levels in Relation to the Extent of Periodontal Disease and Tooth Loss in the Elderly.

The oral innate immune response may diminish with aging. In the present study, the aim was to examine human β-defensin (hBD) 1-3 and human neutrophil peptide (HNP)-1 levels in the saliva of an elderly population to establish the extent of periodontal disease and tooth loss. A total of 175 individuals aged ≥ 65 years were divided into five groups based on the number of teeth with a pocket depth ≥ 4 mm as follows: 17 pocket-free individuals (Control), 55 individuals having 1-6 pocket teeth (PerioA), 33 individuals having 7-13 pocket teeth (PerioB), 29 individuals having at least 14 pocket teeth (PerioC), and 41 edentulous individuals. Their salivary defensin levels were measured with ELISA kits. The salivary HNP-1 levels were significantly higher in the Perio groups (PerioB: p < 0.001 and PerioC: p < 0.001) in comparison to the Control. The associations between salivary HNP-1 levels and the number of pocket teeth remained significant after adjustments for age, gender, level of education, and number of teeth. The salivary HNP and hBD levels differed in terms of their correlation to the extent of periodontal disease and tooth loss in the elderly.

HNP-1 Reverses Hypertensive Left Ventricular Hypertrophy by Inhibiting the NF-кB Signaling Pathway

Background: Human neutrophil peptide-1 (HNP-1) is a commonly investigated therapeutic agent. However, its role in hypertensive left ventricular hypertrophy (HLVH) remains unclear. Methods: We measured HNP-1 levels in patients with hypertension and treated HLVH rat and H9c2 cell hypertrophy models with HNP-1. Cardiomyocyte hypertrophy indexes (i.e., single-cell surface area, left ventricular fibrosis area, BNP levels, and β-MHC levels) were measured with hematoxylin-eosin and Masson’s trichrome staining and WB. NF-кB signaling factors (i.e., IKKβ, p-IKKβ, IкBα, p-IкBα, p65, and p-p65) were measured with WB and qPCR. Finally, inflammatory factors (i.e., IL-6, IL-1α, and TNF-α) were measured with ELISA. Results: HNP-1 levels were lower in the exposure than the control groups (M (95% CI), 48.83 (45.64–52.26) vs. 59.03 (55.62–62.54), P = 0.000). Diminished HNP-1 was associated with HLVH occurrence in patients. HLVH rat and H9c2 cell hypertrophy models revealed elevated cardiomyocyte hypertrophy indexes and NF-кB signaling and inflammatory factors. However, each HNP-1 treatment group showed lower levels of the aforementioned indices than were observed in the model groups. Conclusion: Diminished HNP-1 is a risk factor for HLVH. HNP-1 treatment may reverse HLVH by inhibiting NF-кB signaling pathways.

On the hull and interval numbers of oriented graphs (Brief Announcement)

In this work, for a given oriented graph D, we study its interval and hull numbers, denoted by ⃗in (D) and ⃗hn (D), respectively, in the oriented geodetic, ⃗P3 and ⃗P*3 convexities. This last one, we believe to be formally defined and first studied in this paper, although its undirected version is well-known in the literature.Concerning bounds, for a strongly oriented graph D and the oriented geodetic convexity, we prove that ⃗hn g(D) ≤m(D)-n(D)+2 and that there is at least one such that ⃗hn g(D) =m(D) - n(D). We also determine exact values for the hull numbers in these three convexities for tournaments, which imply polynomial-time algorithms to compute them. These results allow us to deduce polynomial-time algorithms to compute ⃗hn P3(D) when the underlying graph of D is split or cobipartite.Moreover, we provide a meta-theorem by proving that if deciding whether ⃗ing(D) ≤k or ⃗hn g(D) ≤k is NP-hard or W[i]-hard parameterized by k, for some i ϵ Z*+, then the same holds even if the underlying graph of D is bipartite. Next, we prove that deciding whether ⃗hn P3(D) ≤ k or ⃗hn P3* (D) ≤ k is W[2]-hard parameterized by k, even if the underlying graph of D is bipartite; that deciding whether ⃗in P3(D) ≤ k or whether ⃗in P3*(D) ≤ k is NP-complete, and the same for ⃗hn P3*(D) ≤ k even if D has no directed cycles and the underlying graph of D is a chordal bipartite graph; and that deciding whether ⃗in P3(D) ≤ k or whether ⃗in P3*(D) ≤ k is W[2]-hard parameterized by k, even if the underlying graph of D is split.Finally, we also argue that the interval and hull numbers in the ⃗P3 and ⃗P*3 convexities can be computed in polynomial time for directed graphs with underlying graph of bounded tree-width by using Courcelle's theorem.

Pursuing neutrophils: systematic scoping review on blood-based biomarkers as predictors of treatment outcomes in inflammatory bowel disease.

Long-term management of inflammatory bowel diseases (IBD) is challenging and the identification of reliable predictors for treatment outcomes is an unmet need. Neutrophil-related biomarkers have been mainly studied in the feces, but blood analyses have inherent advantages. To review the recent learnings on the ability of blood-based neutrophil-expressed biomarkers to predict treatment outcomes in IBD. Systematic scoping review. We performed a literature search in Pubmed, EMBASE, SCOPUS, Web of Science, ScienceDirect, and Cochrane Central Register of Controlled Trials from inception until May 2022 according to Preferred Reporting Items for Systematic reviews and Meta-Analyses guidelines. All human studies associating blood-based neutrophil-related compounds with the prediction of disease progression, complication onset, or treatment outcomes were included. From 1032 retrieved entries, 34 studies were selected, 32 published in 2013 or later. In all, 17 biomarkers from granules, cytoplasm, plasmatic membrane, and plasma were explored. In total, 1850 Crohn's disease (CD) and 1122 ulcerative colitis non-duplicated patients were included. The most mentioned biomarkers were nCD64, serum calprotectin (SC), oncostatin M (OSM), neutrophil elastase-generated calprotectin fragment (CPa9-HNE), and triggering receptor expressed on myeloid cells 1 (TREM1). Six biomarkers showed promising results: OSM, SC, eNAMPT, nCD64, TREM1, and CPa9-HNE. Variable positive signals were found for human neutrophil peptide 1-3, LL-37, S100A12, and neutrophil gelatinase-associated lipocalin. No predictive ability was found for the remaining markers. Sharing a neutrophil compartment did not indicate similar behavior. Advances in the last decade began to unveil the untapped potential of the readily accessible blood neutrophil-expressed biomarkers, especially nCD64, TREM1, and CPa9-HNE. Current evidence suggests that future research should focus on well-defined subpopulations instead of a one-size-fits-all biomarker. https://osf.io/kes9a.

A proteomic approach to better understand the role of human neutrophil peptides in the NSCLC microenvironment

A proteomic approach to better understand the role of human neutrophil peptides in the NSCLC microenvironment

Differential Gene Expression Analysis for Aromatase Inhibitors Treatment on Breast Cancer Patients

Breast cancer is the most common global malignancy and the leading cause of cancer deaths for women. Aromatase inhibitors are clinically recommended therapy for the disorders. However, resistance to aromatase inhibitors therapies still occurs and the underlined molecular mechanisms are still poorly understood. The tumour specimens consisted of aromatase inhibitor-sensitive and aromatase inhibitor-resistant breast cancer patients, who were compared and the differentially expressed genes for aromatase inhibitor-sensitive patients were generated and related genetic network was explored. The enriched cellular process and signaling cassettes for aromatase inhibitor-sensitive patients were also in-depth investigated. Compared with the aromatase inhibitor-resistant patients, 896 genes showed a significant differential expression. 621 were significantly up-regulated and 275 were remarkably downregulated. From the interaction network, the differentially expressed genes including signal recognition particle 72, B-cell lymphoma-2 associated transcription factor 1, cathelicidin antimicrobial peptide and defensin alpha 1B could be considered as the key target genes for aromatase inhibitors treatment in breast cancer patients. These differentially expressed genes were enriched in processes involving immunoglobulin complex, cell division and others while, drug metabolism and other signalling mechanisms were enriched in aromatase inhibitors treatment of breast cancer patients. The study speculated several novel gene targets for aromatase inhibitors therapeutic treatment of breast cancer patients and initiated a beneficial direction for future aromatase inhibitors drug resistance research.

Wound alpha defensin levels are significantly higher in patients with fracture related infection: A pilot, prospective cohort study

BackgroundIn absence of frank purulence, wound cultures represent ‘gold-standard’ for diagnosis of fracture related infection (FRI). However, these are time-intensive, and may be falsely negative, necessitating the need for accurate and rapid biomarker-based diagnosis. We conducted this study to determine the accuracy of 3 wound-based biomarkers for the diagnosis of FRI. MethodsThis was a prospective cohort study on adult patients who underwent an operative procedure for an upper or lower limb fracture. Wound fluid levels of alpha-defensin (AD), neutrophil elastase (NE) and IL-6 were evaluated on post-operative day 2, and patients were followed up for one month. Patients were categorized as cases (FRI) or controls (no FRI), on the basis of the consensus definition of FRI. Univariate analysis, along with receiver operating characteristic (ROC) analysis was performed. Results48 patients were included. AD levels showed a 2.6-fold elevation in cases (n = 26, Median = 23.74 µg/ml) as compared to controls (n = 22, Median = 8.78 µg/ml). The area under the curve for this variable was 0.71 (95% Confidence Intervals = 0.56 – 0.86). The levels of NE and IL-6 were not significantly different between cases and controls. ConclusionWound AD levels are significantly elevated in patients with FRI. However, these results need to be validated in a larger cohort of patients before it can be used as a biomarker of FRI.

Alpha-defensin as a diagnostic tool in revision total knee arthroplasties with unexpected positive intraoperative cultures and unexpected culture negative intraoperative cultures.

Unexpected-positive-intraoperative-cultures (UPIC) are common in presumed aseptic revision-total-knee-arthroplasties (rTKA). However, the clinical significance is not entirely clear. In contrast, in some presumably septic rTKA, identification of an underlying pathogen was not possible, so-called unexpected-negative-intraoperative-cultures (UNIC). The purpose of this study was to evaluate the potential use of synovial alpha-defensin (AD) levels in these patients. Synovial AD levels from 143 rTKAs were evaluated retrospectively from our prospectively maintained institutional periprostetic joint infection (PJI) biobank and database. The 2018-International Consensus Meeting (ICM) criteria was used to define the study groups. Samples from UPICs with a minimum of one positive intraoperative culture (ICM 2- ≥ 6) (n = 20) and UNIC's (ICM ≥ 6) (n = 14) were compared to 34 septic culture-positive samples (ICM ≥ 6) and 75 aseptic culture-negative (ICM 0-1). Moreover, AD-lateral-flow-assay (ADLF) and an enzyme-linked-immunosorbent-assay (ELISA) in detecting the presence of AD in native and centrifuged synovial fluid specimens was performed. Concentration of AD determined by ELISA and ADLF methods, as well as microbiological, and histopathological results, serum and synovial parameters along with demographic factors were analysed. AD was positive in 31/34 (91.2%) samples from the septic culture-positive group and in 14/14 (100%) samples in the UNIC group. All UPIC samples showed a negative AD result. Positive AD samples were highly associated with culture positive and histopathological results (p < 0.001). No high-virulent microorganisms (0/20) were present in the UPIC group, compared to infected-group (19/34; 55.9%). High virulent microorganisms showed a positive AD result in 89.5% (17/19) of the cases. Methicillin resistant Staphylococcus epidermis (MRSE) infections had significantly higher AD levels than with methicillin susceptible S. epidermdis (MSSE) (p = 0.003). ELISA and ADLF tests were positive with centrifuged (8/8) and native (8/8) synovial fluid. AD showed a solid diagnostic performance in infected and non-infected revisions, and it provided an additional value in the diagnosis of UPIC and UNIC associated to rTKAs. Pathogen virulence as well as antibiotic resistance pattern may have an effect on AD levels. Centrifugation of synovial fluid had no influence on ADLF results.

Are Accuracy Studies for Periprosthetic Joint Infection Diagnosis Inherently Flawed? And What to Do with Schrödinger's Hips? A Prospective Analysis of the Alpha Defensin Lateral-Flow Test in Chronic Painful Hip Arthroplasties.

The most recent diagnostic criteria for periprosthetic joint infection (PJI) include the use of the alpha-defensin (AD) lateral-flow (LF) test, but hip and knee arthroplasties were usually combined in previous studies. This prospective study was designed to examine the accuracy of the AD-LF test for diagnosis of PJI in chronic painful total hip arthroplasties (THA). Patients with chronic painful hip arthroplasties were prospectively enrolled between March 2018 and May 2020. Exclusion criteria included acute PJI or an insufficient amount of synovial fluid. The modified Musculoskeletal Infection Society (MSIS) criteria were primarily used for PJI diagnosis. Fifty-seven patients were included in the analysis group. Revision surgery was not performed in 38 patients, for different reasons (clinical group); these patients remain "Schrödinger's hips": in such cases PJI cannot be excluded nor confirmed until you "open the box". The result of the AD-LF test was positive in nine patients and negative in 48 patients. Six patients were diagnosed with PJI. AD-LF sensitivity (MSIS criteria) was 83% (95% confidence interval [CI] 36-100%) and specificity was 92% (95% CI 81-98%). The positive and negative predictive value were 56% and 98%, respectively. The AD test is useful in addition to the existing arsenal of diagnostic tools, and can be helpful in the decision-making process. Not all patients with chronical painful THA will undergo revision surgery. Consequently, in order to determine the reliable diagnostic accuracy of this test, future PJI diagnostic studies should include a second arm of "Schrödinger's hips".

The Olfactomedin-4-Defined Human Neutrophil Subsets Differ in Proteomic Profile in Healthy Individuals and Patients with Septic Shock

The specific granule glycoprotein olfactomedin-4 (Olfm4) marks a subset (1–70%) of human neutrophils and the Olfm4-high (Olfm4-H) proportion has been found to correlate with septic shock severity. The aim of this study was to decipher proteomic differences between the subsets in healthy individuals, hypothesizing that Olfm4-H neutrophils have a proteomic profile distinct from that of Olfm4 low (Olfm4-L) neutrophils. We then extended the investigation to septic shock. A novel protocol for the preparation of fixed, antibody-stained, and sorted neutrophils for LC-MS/MS was developed. In healthy individuals, 39 proteins showed increased abundance in Olfm4-H, including the small GTPases Rab3d and Rab11a. In Olfm4-L, 52 proteins including neutrophil defensin alpha 4, CXCR1, Rab3a, and S100-A7 were more abundant. The data suggest differences in important neutrophil proteins that might impact immunological processes. However, in vitro experiments revealed no apparent difference in the ability to control bacteria nor produce oxygen radicals. In subsets isolated from patients with septic shock, 24 proteins including cytochrome b-245 chaperone 1 had significantly higher abundance in Olfm4-H and 30 in Olfm4-L, including Fc receptor proteins. There was no correlation between Olfm4-H proportion and septic shock severity, but plasma Olfm4 concentration was elevated in septic shock. Thus, the Olfm4-H and Olfm4-L neutrophils have different proteomic profiles, but there was no evident functional significance of the differences in septic shock.

Study of alpha defensin in coronary artery disease and type 2 diabetes mellitus on the background of metabolic syndrome

Background. The prevalence of metabolic syndrome (MS), a cluster of risk factors for type 2 diabetes mellitus (DM) and coronary artery disease (CAD), is increasing annually. MS is associated with an almost 2-fold increase in the risk of cardiovascular complications and 1.5-fold increase in all-cause mortality. Systemic inflammation is considered to be one of the pathogenetic links in the development of MS. The study of alpha defensins as inducers of inflammation with antimicrobial properties in patients with MS is increasing. However, changes in the serum level of alpha defensin in MS, with or without type 2 DM and CAD, remain insufficiently studied that determines the feasibility of conducting this study. The objective: to evaluate serum levels of alpha defensin in individuals with metabolic syndrome, type2 diabetes mellitus and coronary artery disease. Materials and methods. Fifty-four patients (27 women, 27 men) with MS, type 2 DM and CAD were examined. The research was carried out at the Lviv Regional State Clinical Medical and Diagnostic Endocrinology Center and Lviv Territorial Medical Association “Clinical Hospital of Planned Treatment, Rehabilitation and Palliative Care”, Lviv City Clinical Hospital 5. Patients with MS were divided into 3 subgroups, depending on the presence of type 2 DM and CAD. The first subgroup included 18 individuals with MS without type 2 DM and CAD, the second— 18 people with MS and type 2 DM, the third— 18 patients with MS, type 2 DM and CAD. The control group consisted of 16 healthy people (8 men, 8 women), the employees of the Lviv Territorial Medical Association “Clinical Hospital of Planned Treatment, Rehabilitation and Palliative Care”, Lviv City Clinical Hospital 5. In patients, the level of alpha defensin was determined, as well as blood lipids (high-density lipoproteins (HDL), very low-density lipoproteins (VLDL)), and the correlations between the above values were investigated. Results. The level of alpha defensin and blood lipids were examined in patients with MS, type 2 DM and CAD. The content of alpha defensin in all participants with MS, both with and without type 2 DM and CAD (18.40±0.89, 24.4±1.2, and 29.1±1.1ng/ml), was significantly increased and exceeded the control indicators (3.72±0.36 ng/ml) by 4.95, 6.56, 7.82 times (p&lt;0.05). The highest parameters of alpha defensin were detected in MS combined with type 2 DM and CAD— 29.1±1.1ng/ml that significantly exceeded the parameters in patients with MS and type 2 DM (24.4±1.2 ng/ml) and group of MS without type 2 DM and CAD (18.40±0.89 ng/ml; p&lt;0.05). Values of HDL cholesterol in patients with MS (1.12±0.08 ng/ml), MS and type 2 DM (1.01±0.07 ng/ml), MS, type 2 DM and CAD (0.95±0.05ng/ml) significantly reduced compared to those in the group of healthy individuals (1.40±0.14 ng/ml; р&lt;0.05), but no significant difference was found between individual subgroups (p&gt;0.5). The parameters of VLDL cholesterol were highest in patients with combination of MS, type 2 DM and CAD (1.32±0.05 ng/ml), and significantly exceeded those in patients both with MS and type 2 DM (0.87±0.10 ng/ml), and with MS (0.73±0.06 ng/ml; p&gt;0.05). Comparative analysis was carried out for alpha defensin and blood lipids. A direct strong correlation (r=0.72; р&lt;0.5) was found between alpha defensin and VLDL cholesterol in MS patients. In people with MS, type 2 DM and CAD, a direct strong correlation was also found between alpha defensin and VLDL cholesterol (r=0.65; р&lt;0.5). In the group of MS and type 2 DM, a weak correlation was revealed (r=0.1; р&lt;0.5). Only weak correlations were found between the levels of alpha defensin and HDL cholesterol (r=–0.15, r=–0.11, r=–0.16; р&lt;0.5). Conclusions. A significant increase in the level of alpha defensin was detected in patients with metabolic syndrome, type 2 diabetes mellitus and coronary artery disease. HDL cholesterol values are significantly reduced in all patients with metabolic syndrome, regardless of the presence or absence of type 2 diabetes mellitus and coronary artery disease. VLDL cholesterol parameters, depending on combined pathology, were highest in patients with metabolic syndrome, type 2 diabetes mellitus, and coronary artery disease, and significantly exceeded the levels of VLDL cholesterol in patients with both MS and type 2 diabetes mellitus, and with metabolic syndrome. Correlations were found between alpha defensin indicators and VLDL cholesterol, they were especially expressed in the presence of coronary artery disease and metabolic syndrome.

Comparative analysis of the stability features of human alpha-defensins as candidates for the future COVID-19 therapy through molecular dynamics

Coronavirus 19 (COVID-19) is still a global health issue to date, SARS-CoV-2 is a novel coronavirus that is responsible for this sickness. The receptor-binding domain of the SARS-CoV-2 virus associates with angiotensin-converting enzyme 2 (ACE-2) and allows the virus to enter human cells. Natural peptides such alpha-defensin are thought to attach to the SARS-CoV-2 RBD and prohibit it from engaging with ACE-2. Molecular dynamics simulations using a computational approach are utilized to understand the stability of six alpha-defensin macromolecules using the Gromacs 2016 software. The trajectories formed are then analyzed using VMD 1.9.4 and BIOVIA Discovery Studio 2020 software. Finally, the free energy is estimated using the MM/PBSA method. The alpha-defensins 2 macromolecules were found to have the best stability based on numerous study results (trajectory visualization, RMSD, RMSF, and free energy calculations). As a result, these macromolecules could be used to build new antiviral treatments for COVID-19 infectious disease candidates.

Limited Diagnostic Utility of Alpha Defensin in the Diagnosis of Periprosthetic Joint Infection

Abstract Introduction/Objective A key component of laboratory stewardship is appropriate test utilization. We evaluated the utility of alpha defensin (AD), a synovial fluid biomarker, in the diagnosis of periprosthetic joint infection (PJI). Since PJI remains a diagnostic challenge in the orthopedic community, the Musculoskeletal Infection Society (MSIS) created the PJI score, a point-based criteria to help risk stratify the likelihood of infection. The score includes AD, synovial fluid white blood cell count, synovial fluid percent neutrophils, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP). AD is a specialized test which adds substantial cost to the evaluation of PJI .The purpose of this study was to investigate the diagnostic utility of the PJI score with AD and without AD (routine laboratory testing of serum and synovial fluid). Methods/Case Report Retrospective chart review was completed on 222 patients with suspected PJI identified at Geisinger Health between January 2019 – March 2021. Data was retrieved through Geisinger’s Phenomic and Analytics Data Core and imported into Excel and JMP v.12 for analysis. All available components of MSIS diagnostic criteria were collected for each patient. Results (if a Case Study enter NA) Of the 222 patients with AD testing, 81% of results were negative (n=178) and 19% results were positive (n=44). AD results corresponded with synovial fluid white blood cell count in 96 % of patients (213/ 222). All components of the PJI diagnostic criteria were collected in 69% of the patients (157/222). Diagnostic criteria calculated without AD showed 43% infected (68/157), 37% possibly infected (58/157), and 20% infected (31/157). Diagnostic criteria calculated with AD showed 43% infected (68/157), 35% possible infected (55/157), and 22% infected (34/157). PJI score including AD compared to PJI score excluding AD showed similar correlation (R2 = 0.93). Conclusion It was found that AD testing does not provide substantial diagnostic value in the evaluation of PJI. Synovial fluid cell count was found to directly correlate with alpha defensin suggesting it’s potential use as a surrogate marker for clinical decision making. Diagnostic criteria obtained through routine testing of serum (ESR, CRP) and synovial fluid (WBC, percent neutrophils) provides sufficient data for diagnosis.

Age‐restricted functional and developmental differences of neonatal platelets

BackgroundDevelopmental ontogeny of neonatal thrombopoiesis retains characteristics that are distinct from adults although molecular mechanisms remain unestablished. MethodsWe applied multiparameter quantitative platelet responses with integrated ribosome profiling/transcriptomic studies to better define gene/pathway perturbations regulating the neonatal‐to‐adult transition. A bioinformatics pipeline was developed to identify stable, neonatal‐restricted platelet biomarkers for clinical application. ResultsCord blood (CB) platelets retained the capacity for linear agonist–receptor coupling linked to phosphatidylserine (PS) exposure and α‐granule release, although a restricted block in cross‐agonist activation pathways was evident. Functional immaturity of synergistic signaling pathways was due to younger ontogenetic age and singular underdevelopment of the protein secretory gene network, with reciprocal expansion of developmental pathways (E2F, G2M checkpoint, c‐Myc) important for megakaryocytopoiesis. Genetic perturbations regulating vesicle transport and fusion (TOM1L1, VAMP3, SNAP23, and DNM1L) and PS exposure and procoagulant activity (CLCN3) were the most significant, providing a molecular explanation for globally attenuated responses. Integrated transcriptomic and ribosomal footprints identified highly abundant (ribosome‐protected) DEFA3 (encoding human defensin neutrophil peptide 3) and HBG1 as stable biomarkers of neonatal thrombopoiesis. Studies comparing CB‐ or adult‐derived megakaryocytopoiesis confirmed inducible and abundant DEFA3 antigenic expression in CB megakaryocytes, ~3.5‐fold greater than in leukocytes (the most abundant source in humans). An initial feasibility cohort of at‐risk pregnancies manifested by maternal/fetal hemorrhage (chimerism) were applied for detection and validation of platelet HBG1 and DEFA3 as neonatal thrombopoiesis markers, most consistent for HBG1, which displayed gestational age‐dependent expression. ConclusionsThese studies establish an ontogenetically divergent stage of neonatal thrombopoiesis, and provide initial feasibility studies to track disordered fetal‐to‐adult megakaryocytopoiesis in vivo.

Антимікробні пептиди (HNPs 1-3 та LL-37) як біомаркери активності запального процесу в дітей, хворих на H. pylori-асоційовану виразку дванадцятипалої кишки

Various gastrointestinal and extragastrointestinal diseases are associated with H. pylori in children and adolescents, but the strongest recommendations for testing and treatment are made only in children and adolescents with duodenal ulcer (DU). The inability of natural immune mechanisms to recognize and eliminate H. pylori leads to the development of acute inflammation. The most promising developments so far are studies of the antibacterial effect of endogenous antimicrobial peptides (AP), among which defensines 1-3 (human neutrophil peptides, HNPs 1-3) and cathelicidins (LL-37) are the most important for the human body. Purpose - to investigate the concentration of HNPs 1-3 and LL-37 in the blood of children with DU in order to determine the activity of the inflammatory process of the mucous membrane. Materials and methods. 65 children aged 7-18 years, suffering from DU and 25 healthy children of the corresponding age (comparison group) were examined. The level of HNPs 1-3 and LL-37 was determined in blood plasma by enzyme-linked immunosorbent assay according to the manufacturers instructions. Results. Patients were divided by age, sex, location and size of the ulcer, presence of the H. pylori bacterium. A toxigenic strain of H. pylori was detected in 80.9% of examined children, and the level of HNPs 1-3 in blood plasma was 3 times higher and the level of LL-37 was 2.5 times higher in children with H. pylori-associated DU than in healthy children (p=0.01). Concentrations of HNPs 1-3 and LL-37 in blood plasma were higher in patients with an active inflammatory process in the mucous membrane and were positively correlated with the degree of inflammation activity (r=0.67, p=0.05 and r=0.69, p=0.01). After eradication therapy, AP levels decrease, while the degree of decrease directly depends on the activity of the inflammatory process. Conclusions. Probably higher concentrations of HNPs 1-3 and LL-37 were found in the blood plasma of children with H. pylori-associated DU. Considering the direct correlations between the levels of HNPs 1-3 and LL-37 and the degree of activity of the inflammatory process, these indicators can be used as biomarkers of the adverse course of H. pylori-associated DU. The research was carried out in accordance with the principles of the Helsinki Declaration. The study protocol was approved by the Local Ethics Committee of all participating institutions. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.