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ObjectivesThis study aimed to define the antimicrobial peptide (AMP) expression pattern of the equine ocular surface and amniotic membrane using a targeted qPCR approach and 3'Tag-sequencing. It will serve as a reference for future studies of ocular surface innate immunity and amniotic membrane therapies.ProceduresA targeted qPCR approach was used to investigate the presence of orthologs for three of the most highly expressed beta-defensins (DEFB1, DEFB4B, and DEFB103A) of the human ocular surface and amniotic membrane in equine corneal epithelium, conjunctiva, and amniotic membrane. 3'Tag-sequencing was performed on RNA from one sample of corneal epithelium, conjunctiva, and amniotic membrane to further characterize their AMP expression.ResultsEquine corneal epithelium, conjunctiva, and amniotic membrane expressed DEFB1, DEFB4B, and DEFB103A. DEFB103A was expressed at the highest amounts in corneal epithelium, while DEFB4B was most highly expressed in conjunctiva and amniotic membrane. 3'Tag-sequencing from all three tissues confirmed these findings and identified expression of five additional beta-defensins, 11 alpha-defensins and two cathelicidins, with the alpha-defensins showing higher normalized read counts than the beta-defensins.ConclusionsThis study identified AMP expression in the equine cornea and conjunctiva, suggesting that they play a key role in the protection of the equine eye, similar to the human ocular surface. We also determined that equine amniotic membrane expresses a substantial number of AMPs suggesting it could potentiate an antimicrobial effect as a corneal graft material. Future studies will focus on defining the antimicrobial activity of these AMPs and determining their role in microbial keratitis.

The class I major histocompatibility (MHC-I) complex is a set of diverse cell surface receptors encoded by the human leukocyte antigen gene complex. These receptors present intracellular antigens to cytotoxic T cells providing information on the state and health of cells. Changes in the immunopeptidome during cancer may provide novel targets for therapeutic intervention. To understand how the tumor immunopeptidome is altered, we developed a mass spectrometry (MS) based platform for isolating and identifying MHC-I peptide antigens in lung tumors. In the course of our work, we encountered several large unknown peptide contaminants which had not been previously reported. To understand the source of these major contaminants, we isolated them using offline fractionation and identified them by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as members of the host defense protein family known as the defensins. To mitigate their detrimental effects, we modified our "Original" data-dependent acquisition (DDA) MS method to narrowly target the MHC-I peptides based on their physical properties including charge state and molecular weight ("z state" DDA), evaluated field asymmetric ion mobility spectrometry to attempt gas-phase separation prior to MS analysis, and developed an immunodepletion approach using defensin specific antibodies. This modified approach improves peptide identification and reduces the impact of defensin contamination in lung tissue samples.

Graphic abstract The coronavirus (COVID-19) pandemic is still spreading all over the world. As reported, angiotensin-converting enzyme-2 (ACE2) is a receptor of SARS-CoV-2 spike protein that initializes viral entry into host cells. Previously, the human defensin 5 (HD5) has been experimentally confirmed to be functional against the SARS-CoV-2. The present study proposes a human cathelicidin known as LL37 that strongly binds to the carboxypeptidase domain of human ACE2 compared to HD5. Therefore, LL37 bears a great potential to be tested as an anti-SARS-CoVD-2 peptide. We investigated the molecular interactions formed between the LL37 and ACE2 as well as HD5 and ACE2 tailed by their thermodynamic stability. The MM-PBSA and free energy landscape analysis outcomes confirmed its possible inhibitory effect against the SARS-CoV-2. The results obtained here could help propose a promising therapeutic strategy against the havoc caused by SARS-CoV-2 infections. Supplementary Information The online version contains supplementary material available at 10.1007/s12539-021-00462-3.

Angira Dasgupta,1– 3 Rahul Chakraborty,1,2 Bodhisattwa Saha,1 Himanshi Suri,1 Praveen Singh,1,2 Anurag Raj,1,2 Bhupesh Taneja,1,2 Debasis Dash,1,2 Shantanu Sengupta,1,2 Anurag Agrawal1,2 1Institute of Genomics and Integrative Biology, New Delhi, India; 2Academy of Scientific and Innovative Research, CSIR-HRDG, Ghaziabad, Uttar Pradesh, 201002, India; 3B R Singh Hospital and Centre for Medical Education and Research, Kolkata, West Bengal, IndiaCorrespondence: Angira DasguptaB R Singh Hospital and Centre for Medical Education and Research, Sealdah, Kolkata, West Bengal, IndiaTel +91 9831517266Email angiradasgupta@gmail.comBackground: Chronic mucous hypersecretion (CMH or chronic bronchitis) per se or when associated with chronic inflammatory airway diseases such as asthma or chronic obstructive pulmonary disease (COPD) has several adverse clinical consequences. The sputum fluid phase has several candidate proteins including mucins which have the potential of being therapeutic targets, but has not yet been explored in-depth. This study aimed at exploring the profile of sputum proteins in various airway diseases.Methods: Sputum from thirty-one patients with various airway diseases was collected and the fluid phase analyzed by LC-MS/MS and subsequently by sequential window acquisition of all theoretical fragments ion spectra (SWATH) (n = 15) for protein quantitation. Hierarchical clustering and functional grouping were performed.Results: A total of 185 proteins were quantitated by SWATH of which 21 proteins were identified which could distinguish between the clinical phenotypes by hierarchical clustering. Functional protein clustering revealed 4 groups: those that are inflammation related, oxidative stress related, mucin related and a cytoskeletal and calcium related group. The levels of eight proteins (Azurocidin1, Neutrophil defensin 3, Lactotransferrin, Calmodulin 3, Coronin1A, Mucin 5B, Mucin 5AC and BPI fold containing family B1) were significantly altered (relative to mean) in exacerbator prone subjects compared to nonexacerbators. Another simple but useful metric which emerged from this study was total protein concentration in sputum which was significantly higher in frequent exacerbators.Conclusion: Sputum proteins can detect the various airway disease clinical phenotypes. Total protein concentration and eight other proteins are biomarkers for frequent exacerbators. The clinical and therapeutic implications of the functional groups of proteins need further evaluation.Keywords: proteomics, chronic bronchitis, asthma, COPD, mucin, phenotyping

BackgroundSystemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and lung as well as involvement of kidney, gastrointestinal system and heart. Aetiology and exact mechanism of disease is poorly understood. The association between antimicrobial peptides (AMPs) and other diseases such as idiopathic pulmonary fibrosis, diffuse panbronchiolitis, pulmoner alveolar proteinosis and psoriasis have been reported. A small number of studies have examined the role of AMPs on autoimmune diseases which has not been studied in scleroderma yet. We aimed to investigate AMP serum levels and their association with disease characteristics of SSc.MethodsForty-two patients (40 female, mean age 42 years) and 38 healthy subjects (32 female, mean age 38 years) were enrolled. For SSc patients, the following data were recorded: disease subset (limited/diffuse), autoantibodies (antinuclear, anti-centromere (ACA), and anti-SCL-70), blood tests, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP), modified Rodnan skin score, presence and history of digital ulcers, kidney, gastrointestinal disease and lung involvement assessed by computed tomography and pulmonary function tests. Association between serum AMPs and disease characteristics were analysed.ResultsTwenty-nine of the patients had diffuse (69%) and 13 of the patients had limited (31%) systemic sclerosis. Average disease duration was 5.5 years. Pulmonary involvement was detected in 20 patients (47.6%). Serum concentration of alpha defensin was higher than healthy subjects (563 ± 415 vs 377 ± 269 ng/mL,p = 0.02). However, no difference was observed for beta-1 and beta-2 defensins in SSc patients and healthy controls. In sub-group analysis patients with interstitial lung disease had higher levels of alpha defensin than those without lung involvement (684 ± 473 vs 430 ± 299 ng/ml,p = 0.04). There was also correlation between alfa defensin serum concentrations and CRP (r = 0.34).ConclusionsAlpha defensin levels are increased in scleroderma patients and correlated with lung involvement indicating a role in the pathogenesis of disease.Trial registrationThis study is not a clinical trial study.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) gains access to host cells by attaching to angiotensin-converting enzyme 2 (ACE2). Vitamin D (VitD) can upregulate ACE2 and has an antagonistic effect on Renin, which exerts a vasodilatation and anti-inflammatory effect against coronavirus disease (COVID-19). However, it may also facilitate viral entry by increasing ACE2 as the main SARS-CoV-2 receptor and mediates ROS production through NADPH oxidase, as a double-edged sword effect. Lung function and the immune system are also influenced by VitD through several mechanisms, including increased natural antibiotics (Defensin and Cathelicidin) and upregulated transforming growth factor-β. A higher IgA, Th2/Th1 ratio, and T-regulatory cells are attributable to VitD effects on the immune cells, while these changes may also be a double-edged sword in COVID-19. Although VitD supplementation might be highly recommended in COVID-19, the administration's dosage and route could be challenging. Furthermore, this issue has not been mentioned in various studies so far. So, the report aimed to explain the current challenges with the application of VitD in COVID-19.

The human ocular surface produces highly conserved cationic peptides. Human β‑defensins (HBDs) serve an important role in innate and adaptive immunity. They are primarily expressed in epithelial cells in response to infection and provide the first line of defence against invading microbes. Defensin β1 (DEFB1) is constitutively expressed and regulated by inflammatory mediators including interferon‑γ, lipopolysaccharide and peptidoglycans. DEFB4A is locally induced in response to microbial infection while DEFB109 is induced via Toll‑like receptor 2. The present study examined the expression of the HBD DEFB1, DEFB4A and DEFB109 genes in pterygium. The pterygium tissues and normal conjunctiva samples were obtained from 18 patients undergoing pterygium surgery. The reverse transcription‑quantitative polymerase chain reaction method was employed to determine the expression of DEFB1, DEFB4A and DEFB109 genes. The results revealed that the expression of DEFB1 and DEFB4A was significantly higher and upregulated in pterygium samples when compared with normal conjunctiva samples from each patient (P

Clinical fungal infections always cause a negative impact on human health. Moreover, during the interaction of pathogenic fungi with the environment and host, many biologically active substances are produced. Here, we report a new toxin-like defensin of purlisin derived from a clinical pathogenic isolate of Purpureocillium lilacinum. The analysis of its genomic and mRNA sequences revealed an open reading frame of 444 bp without introns. The deduced precursor peptide was composed of 147 amino acids, and the mature peptide were identified at protein level by LC-ESI-Q-TOF-MS/MS. After posttranslational processing, the precursor peptide of purlisin was split into two independent peptides. The two mature defensins, purlisin-NT and purlisin-CT, are consisting of 36 and 38 amino acid residues, which can form three and four intramolecular disulfide bonds, respectively. The results of circular dichroism and homology modeling revealed that they adopted a representative cysteine-stabilized α-helical and β-sheet motif. The purlisin-NT showed a dose-dependent selective inhibition of immune-related hKv1.3 target channel with IC50 value of 0.2 ± 0.04 μM but no obvious antibacterial activity, while the purlisin-CT displayed antimicrobial activities against gram-positive bacteria as well as clinical isolates of MRSA and low affinities for potassium channels. Our findings suggest that purlisin-NT with immunosuppressive effects and purlisin-CT possessing antibacterial activities are adapted to the survival and pathogenicity of clinical P lilacinumis. Moreover, they can also be used as templates for the design of novel antibacterial peptide and immunosuppressive agents.

Kawasaki disease (KD) usually affects the children younger than 5 years of age and subsequently causes coronary artery lesions (CALs) without timely identification and treatment. Developing a robust and fast prediction method may facilitate the timely diagnosis of KD, significantly reducing the risk of CALs in KD patients. The levels of inflammatory serum proteins dramatically vary during the onsets of many immune diseases, including in KD. However, our understanding of their pathogenic roles in KD is behind satisfaction. The purpose of this study was to evaluate candidate diagnostic serum proteins and the potential mechanism in KD using iTRAQ gel-free proteomics. We enrolled subjects and conducted iTRAQ gel-free proteomics to globally screen serum proteins followed by specific validation with ELISA. Further in vitro leukocyte trans-endothelial model was also applied to investigate the pathogenesis roles of inflammatory serum proteins. We identified six KD protein biomarkers, including Protein S100-A8 (S100A8), Protein S100-A9 (S100A9), Protein S100-A12 (S100A12), Peroxiredoxin-2 (PRDX2), Neutrophil defensin 1 (DEFA1) and Alpha-1-acid glycoprotein 1 (ORM1). They enabled us to develop a high-performance KD prediction model with an auROC value of 0.94, facilitating the timely identification of KD. Further assays concluded that recombinant S100A12 protein treatment activated neutrophil surface adhesion molecules responsible for adhesion to endothelial cells. Therefore, S100A12 promoted both freshly clinically isolated neutrophils and neutrophil-like cells to infiltrate through the endothelial layer in vitro. Finally, the antibody against S100A12 may attenuate the infiltration promoted by S100A12. Our result demonstrated that evaluating S100A8, S100A9, S100A12, PRDX2, DEFA1 and ORM1 levels may be a good diagnostic tool of KD. Further in vitro study implied that S100A12 could be a potential therapeutic target for KD.

Background Although estrogen deficiency has been proposed as a risk factor for oral mucosal inflammatory diseases in post-menopausal women, the mechanisms involved remain unclear. This study aimed to investigate the effect of 17β-estradiol (E2) on the inflammatory response stimulated by interleukin-1 beta (IL-1β) in human oral mucosal epithelial cells (hOMECs) and its possible mechanism. Methods Primary hOMECs were obtained from female infants and cultured in keratinocyte growth medium. The hOMECs at second passage were collected and stimulated by 10-7 mol/L ICI182,780 or 10-7 mol/L G1 for 1 hour, E2 (10-7 mol/L, 10-8 mol/L, 10-9 mol/L) for 36 hour, 100 ng/mL IL-1β for 12 hours, respectively. Human beta-2 defensin (hBD-2), tumor necrosis factor-alpha (TNF)-α, IL-6, IL-8, estrogen receptor-alpha (ERα), estrogen receptor-beta (ERβ), and G protein-coupled receptor 30 (GPR30) mRNA levels and protein levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), and Western Blot (WB), respectively. Results Expression of hBD-2 and inflammatory cytokines increased after IL-1β stimulation, which was down-regulated by E2 pre-treatment. With ICI182,780, the suppression of E2 on hBD-2 mRNA was attenuated. With G1, the mRNA expression and protein expression of hBD-2 were reduced. Conclusion Pre-treatment of hOMECs with E2 at physiological concentrations inhibited the IL-1β-induced expression of hBD-2 and inflammatory cytokines. The protective effects of E2 suggest its potential use treating oral inflammatory diseases in clinical practice.

The human pathogenic bacterium Clostridioides difficile produces two exotoxins TcdA and TcdB, which inactivate Rho GTPases thereby causing C. difficile-associated diseases (CDAD) including life-threatening pseudomembranous colitis. Hypervirulent strains produce additionally the binary actin ADP-ribosylating toxin CDT. These strains are hallmarked by more severe forms of CDAD and increased frequency and severity. Once in the cytosol, the toxins act as enzymes resulting in the typical clinical symptoms. Therefore, targeting and inactivation of the released toxins are of peculiar interest. Prompted by earlier findings that human α-defensin-1 neutralizes TcdB, we investigated the effects of the defensin on all three C. difficile toxins. Inhibition of TcdA, TcdB, and CDT was demonstrated by analyzing toxin-induced changes in cell morphology, substrate modification, and decrease in transepithelial electrical resistance. Application of α-defensin-1 protected cells and human intestinal organoids from the cytotoxic effects of TcdA, TcdB, CDT, and their combination which is attributed to a direct interaction between the toxins and α-defensin-1. In mice, the application of α-defensin-1 reduced the TcdA-induced damage of intestinal loops in vivo. In conclusion, human α-defensin-1 is a specific and potent inhibitor of the C. difficile toxins and a promising agent to develop novel therapeutic options against C. difficile infections.

Antimicrobial peptides are considered to be one of the candidate antimicrobial agents for antibiotic-resistant bacterial infection in the future. The effects of antimicrobial peptide hBD3-CBD on Pseudomonas aeruginosa PA14 and PA14 ΔexsA were analyzed by the bactericidal effects, hemolysis assays, pyocyanin pigment productions, and virulence factor expressions (exoU, exoS, hcnA, and lasB). Pyocyanin production and virulence factor expressions are important features of the type III secretion system in Pseudomonas aeruginosa. HBD3-CBD killed PA14 and PA14 ΔexsA with similar efficiency; it lowered the hemolysis levels of PA14 and PA14 ΔexsA and reduced the pyocyanin production, biofilm formation, and exoU, exoS, and lasB expressions in PA14. Compared with PA14, PA14 ΔexsA showed a lower hemolysis effect, pyocyanin production, exoU, and lasB expressions. The effects of hBD3-CBD on the PA14 toxin secretion were similar to the changes in the type III secretion system mutant isolate PA14 ΔexsA. Our results demonstrated that the type III secretion system was involved in the biological functions on PA 14 from hBD3-CBD.

The objective of this study was to evaluate expression of a cluster of genes encoding β-defensin antimicrobial peptides in neutrophils of postpartum cows in relation to prepartum dietary cation-anion difference (DCAD), vitamin D, and postpartum disease. Pregnant dry Holstein cows (28 nulliparous and 51 parous) at 255 d gestation were blocked by parity and randomly assigned to 4 prepartum diets of positive (+130 mEq/kg) or negative (-130 mEq/kg) DCAD and either 3 mg vitamin D3 or 3 mg of 25-hydroxyvitamin D3 per 11 kg of dry matter/d. Treatment diets were fed from 255 d of gestation until calving. Peripheral blood neutrophils of 35 parous cows were collected at 0 and 3 d after calving and stimulated with 0 or 100 ng/mL of lipopolysaccharide (LPS). Furthermore, serum Ca and incidences of postpartum diseases were recorded for all cows. The mRNA transcripts of β-defensin genes were quantified by real-time PCR, and data were analyzed with a general linear mixed model to test for fixed effects and interactions of day, level of DCAD, source of vitamin D, and incidence of disease. Effects of DCAD and vitamin D on neutrophil oxidative burst and phagocytosis were previously reported but were analyzed for effects of disease in the present study. Transcripts for DEFB1, DEFB3, DEFB4, DEFB5, DEFB7, DEFB10, and lingual antimicrobial peptide (LAP) in neutrophils were upregulated by LPS at 0 d but not at 3 d. Transcripts for DEFB4 and DEFB7 in LPS-stimulated neutrophils were greater in cows fed negative DCAD diets compared with positive DCAD. Source of vitamin D (vitamin D3 vs. 25-hydroxyvitamin D3) did not affect expression of β-defensins in neutrophils. Cows with postpartum subclinical hypocalcemia (serum Ca

Defensins form an integral part of the cationic host defence peptide (HDP) family, a key component of innate immunity. Apart from their antimicrobial and immunomodulatory activities, many HDPs exert multifaceted effects on tumour cells, notably direct oncolysis and/or inhibition of tumour cell migration. Therefore, HDPs have been explored as promising anticancer therapeutics. Human β-defensin 2 (HBD-2) represents a prominent member of human HDPs, being well-characterised for its potent pathogen-killing, wound-healing, cytokine-inducing and leukocyte-chemoattracting functions. However, its anticancer effects remain largely unknown. Recently, we demonstrated that HBD-2 binds strongly to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2), a key mediator of defensin-induced cell death and an instructional messenger during cell migration. Hence, in this study, we sought to investigate the lytic and anti-migratory effects of HBD-2 on tumour cells. Using various cell biological assays and confocal microscopy, we showed that HBD-2 killed tumour cells via acute lytic cell death rather than apoptosis. In addition, our data suggested that, despite the reported PI(4,5)P2 interaction, HBD-2 does not affect cytoskeletal-dependent tumour cell migration. Together, our findings provide further insights into defensin biology and informs future defensin-based drug development.

The term host defense peptides arose at the beginning to refer to those peptides that are part of the host’s immunity. Because of their broad antimicrobial capacity and immunomodulatory activity, nowadays, they emerge as a hope to combat resistant multi-drug microorganisms and emerging viruses, such as the case of coronaviruses. Since the beginning of this century, coronaviruses have been part of different outbreaks and a pandemic, and they will be surely part of the next pandemics, this review analyses whether these peptides and their derivatives are ready to be part of the treatment of the next coronavirus pandemic.

Antimicrobial peptides (AMPs) are short cationic amphipathic peptides with a wide range of antimicrobial properties and play an important role in the maintenance of immune homeostasis by modulating immune responses in the reproductive tract. As intra-amniotic infection and microbial dysbiosis emerge as common causes of preterm births (PTBs), a better understanding of the AMPs involved in the development of PTB is essential. The altered expression of AMPs has been reported in PTB-related clinical presentations, such as preterm labor, intra-amniotic infection/inflammation, premature rupture of membranes, and cervical insufficiency. Moreover, it was previously reported that dysregulation of AMPs may affect the pregnancy prognosis. This review aims to describe the expression of AMPs associated with PTBs and to provide new perspectives on the role of AMPs in PTB.

American oyster defensin (AOD) was previously purified from acidified gill extract of the American oyster, Crassostrea virginica. AOD is composed of 38 amino acids with three disulfide bonds and exhibits strong antimicrobial activity against Gram-positive bacteria as well as significant activity against Gram-negative bacteria. Here, to develop promising peptides into antibiotic candidates, we designed five arginine-rich analogs (A0, A1, A2, A3, and A4), predicted their loop and extended strand/random structures—including nine amino acids and a disulfide bond derived from the C-terminus of AOD—and described their antimicrobial and cytotoxic effects, as well as their modes of action. In our experimental results, the A3 and A4 analogs exhibited potent antimicrobial activity against all test organisms—including four Gram-positive bacteria, six Gram-negative bacteria, and Candida albicans—without cell toxicity. A sequence of experiments, including a membrane permeabilization assay, DNA binding study, and DNA polymerization inhibition test, indicated that the two analogs (A3 and A4) possibly&nbsp, did not act directly on the bacterial membrane but instead interacted with intracellular components such as DNA or DNA amplification reactions. AOD analogs also showed strong bacterial inhibition activity in the plasma environment. In addition, analog-treated microbial cells clearly exhibited membrane disruption, damage, and leakage of cytoplasmic contents. Collectively, our results suggest that two analogs, A3 and A4, have potent antimicrobial activity via DNA interaction and have the potential for development into novel antimicrobial agents.

Anti-microbial peptides (AMPs), small biologically active molecules, produced by different organisms through their innate immune system, have become a considerable subject of interest in the request of novel therapeutics. Most of these peptides are cationic-amphipathic, exhibiting two main mechanisms of action, direct lysis and by modulating the immunity. The most commonly reported activity of AMPs is their anti-bacterial effects, although other effects, such as anti-fungal, anti-viral, and anti-parasitic, as well as anti-tumor mechanisms of action have also been described. Their anti-parasitic effect against leishmaniasis has been studied. Leishmaniasis is a neglected tropical disease. Currently among parasitic diseases, it is the second most threating illness after malaria. Clinical treatments, mainly antimonial derivatives, are related to drug resistance and some undesirable effects. Therefore, the development of new therapeutic agents has become a priority, and AMPs constitute a promising alternative. In this work, we describe the principal families of AMPs (melittin, cecropin, cathelicidin, defensin, magainin, temporin, dermaseptin, eumenitin, and histatin) exhibiting a potential anti-leishmanial activity, as well as their effectiveness against other microorganisms.

The objective of this study was to identify proteins that are differentially expressed in the cystic wall tissues of ovarian endometriotic cysts, simple ovarian cysts, and in normal ovarian tissues. Specimens of ovarian endometriotic cyst wall tissue, simple ovarian cyst wall tissue, and normal ovarian tissue (six specimens per group) were collected from patients who received gynecologic surgery, respectively. Differentially expressed proteins related to the ovarian endometriotic cysts were screened by use of isobaric tags for relative and absolute quantitation (iTRAQ) combined with functional annotation and bioinformatics analyses. All differentially expressed proteins related to cysts were validated using immunohistochemistry methods in recurrent and non-recurrent ovarian endometriotic cyst. A total of 359 proteins were identified as up-regulated in ovarian endometriotic cyst groups when compared with both the normal ovary and simple ovarian cyst groups. The levels of 27 proteins were >two-fold higher in the ovarian endometriotic cyst group than that in the other two groups. Of note, the five most significantly upregulated proteins were Charcot-Leyden Crystal Galectin (CLC), Defensin, alpha 1 (DEFA1), S100 calcium-binding protein A9 (S100A9), S100 calcium-binding protein A8 (S100A8), and Ferritin Light Chain (FTL). Immunohistochemistry results showed that the changes of S100A9 and S100A8 were consistent with the results shown by iTRAQ. However, no similarity of CLC, DEFA1, and FTL proteins was found between iTRAQ and immunohistochemistry. The ratio of patients with abnormally high S100A9 and S100A8 expression in the recurrent ovarian endometriotic cyst group was significantly higher than that in the non-recurrent group (P < 0.05). Our data identify differentially expressed proteins S100A9 and S100A8, and suggest they may serve as novel molecular markers to predict postoperative recurrence of an ovarian endometriotic cysts.Abbreviations: iTRAQ: isobaric tags for relative and absolute quantitation; HPRD: Human Protein Reference Database; GO: Gene Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; EM: Endometriosis; COX-2: cyclooxyenase-2; NF-kB: nuclear factor kappa-B; PR-B: progesterone receptor type B.

Streptococcus pneumoniae (S. pneumoniae) is the most common bacterial cause of community-acquired pneumonia. Increasing rates of antibiotic-resistant S. pneumoniae strains impair therapy and necessitate alternative treatment options. In this study, we analysed insect-derived antimicrobial peptides (AMPs) for antibacterial effects on S. pneumoniae in a human in vitro infection model. AMP effects on bacterial growth were examined by colony forming unit (CFU)-assays, and growth curve measurements. Furthermore, cytotoxicity to primary human macrophages was detected by measuring lactate-dehydrogenase release to the supernatant. One AMP (Defensin 1) was tested in a model of primary human monocyte-derived macrophages infected with S. pneumoniae strain D39 and a multi-resistant clinical isolate. Inflammatory reactions were characterised by qPCR and multiplex-ELISA. In total, the antibacterial effects of 23 AMPs were characterized. Only Tribolium castaneum Defensin 1 showed significant antibacterial effects against S. pneumoniae strain D39 and a multi-resistant clinical isolate. During in vitro infection of primary human macrophages with S. pneumoniae D39, Defensin 1 displayed strong antibacterial effects, and consequently reduced bacteria-induced cytokine expression and release. In summary, Tribolium castaneum Defensin 1 showed profound antibacterial effectivity against Streptococcus pneumoniae D39 and a multi-resistant clinical isolate without unwanted cytotoxic or inflammatory side effects on human blood-derived macrophages.

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in 1 H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.

Myeloid-derived suppressor cells (MDSCs) are present in the human lung microenvironment, and they may be involved in the local inflammatory process in chronic obstructive pulmonary disease (COPD). Chronic inflammation in COPD may induce immunogenic cell death of structural airway cells, causing the release of damage-associated molecular patterns (DAMPs). DAMPs may activate the innate and adaptive immune system. The relationship between MDSCs and DAMPs in COPD is poorly described in the available literature.Objectives. (1) Assessment of MDSC percentage and DAMP concentration in bronchoalveolar lavage fluid (BALF) and peripheral blood. (2) Analysis of the relationship between MDSC percentage and chosen DAMPs.Patients and Methods. 30 COPD patients were included. Using monoclonal antibodies directly conjugated with fluorochromes in flow cytometry, MDSCs were assessed in BALF and peripheral blood. The concentration of DAMPs was estimated using sandwich ELISA. Using the Bradford method, the total protein concentrations were evaluated.Results. The percentage of MDSCs among MC in BALF correlated well with the concentration of defensin and heat shock protein 27. Assessing the percentage of MDSCs among all leukocytes in BALF, we revealed a significant correlation with the concentration of defensin, hyaluronic acid, and surfactant protein A. No dependencies occurred between DAMPs and MDSCs in peripheral blood.Conclusion. MDSCs and DAMPs occur in the COPD patient lung microenvironment. Significant correlations between them found in BALF may indicate their influence on the local inflammatory process in COPD. These relationships allow better understanding of the inflammatory process in COPD.

Background Candida albicans (C albicans) is the most common fungal pathogen causing opportunistic infections. IL17 (IL17A) is a vital mediator of antifungal immunity. The aim of the study was to investigate the effect of recombinant human interleukin 17A (rhIL17A) on human oral mucosal epithelial cells (hOMECs) defending against C albicans infection. Methods Human oral mucosal epithelial cells were divided into four groups: C albicans+ (MOI = 0.1), rhIL17A+ (100 μg/L), rhIL17A + C albicans+ (MOI = 0.1, rhIL17A:100 μg/L) and blank control. Then, C albicans growth was observed after 24 hours. Human beta-2 defensin (hBD-2), S100A8 and LL-37 in supernatants and their mRNAs in cells were measured by enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction, respectively. Results In C albicans+ group, C albicans hyphae formation and the death of infected hOMECs were observed. However, in the rhIL17A + C albicans+ group, IL17 inhibited both hypha formation, and C albicans from infecting hOMECs and its further growth. There was no statistical significance in adhesion rates of C albicans to hOMECs. Compared with the control group, the level of hBD-2 mRNA has increased, while hBD-2 and hBD-2 mRNA levels in the rhIL17A + C albicans+ group were the highest. Both hBD-2 and hBD-2 mRNA levels were higher in the rhIL17A+ group than in the C albicans+ group. S100A8 and LL-37 mRNAs have similar trend, and both upregulated after treatment with rhIL17A; however, protein levels were undetectable. Conclusion Recombinant human interleukin 17A may inhibit C albicans from infecting hOMECs by affecting the growth and reproduction of C albicans as well as the formation of hyphae. Besides, rhIL17A might induce hBD-2, S100A8 and LL-37 secretion from hOMECs to strengthen their anti-infective ability.

Platelets are anucleate cells that have a role in several innate immune functions, including the secretion of proteins with antimicrobial activity. Several studies have demonstrated the ability of platelets to secrete thrombin-induced platelet microbicidal proteins and antimicrobial peptides, like hBD-1. However, the expression and secretion of defensins of the alpha family by platelets have not been fully elucidated. The aim of this study was to characterize the expression of defensin alpha 1 (DEFA1) in human platelets and megakaryocytes. Our data indicate that DEFA1 mRNA and protein are present in peripheral blood platelets and in the megakaryoblastic leukemia cell line (MEG-01). DEFA1 co-localize with α-granules of platelets and MEG-01 cells, and was also detected in cytoplasm of MEG-01 cells. The assay of our in vitro model of platelet-like particles (PLPs) revealed that MEG-01 cells could transfer DEFA1 mRNA to their differentiated PLPs. Furthermore, platelets secreted DEFA1 into the culture medium when activated with thrombin, adenosine diphosphate, and lipopolysaccharide; meanwhile, MEG-01 cells secreted DEFA1 when activated with thrombopoietin. Platelet's secreted DEFA1 can rebind to platelet's surface and have antibacterial activity against the gram-negative bacteria Escherichia coli. In summary, our data indicate that both, human platelets and megakaryocytes, can express and secrete DEFA1. These results suggest a new role of platelets and megakaryocytes in the innate immune response.

Purpose This study aimed to clarify the influence of chewing on human β-defensin 2 (hBD-2) and secretory immunoglobulin A (SIgA) expression levels. Methods We included 15 healthy males with no missing teeth (mean age, 25.5 ± 2.5 years). Subjects were instructed to chew a piece of gum for 30 min. Saliva and skin-extraction samples were collected before and after chewing for 15 and 30 min. hBD-2 and SIgA concentrations in the samples were determined using enzyme-linked immunosorbent assay (ELISA). hBD-2 and SIgA expression levels before and after chewing were analyzed using the Mann–Whitney U test, following the Friedman test. The significance level was 0.05. Results The hBD-2 level in skin-extraction samples was significantly different before (99.4 ± 17.3 pg/mL) and after chewing for 30 min (142 ± 23.0 pg/mL). The SIgA level in skin-extraction samples was also significantly different before (2.39 ± 0.25 μg/mL) and after chewing for 30 min (3.61 ± 0.33 μg/mL). No significant difference was noted in either hBD-2 or SIgA secretion rate in saliva between before and after chewing. Conclusions Chewing gum for 30 min increased hBD-2 and SIgA expression levels in skin. Moreover, chewing gum could influence the secretion pattern of these two biomolecules on skin, but not in saliva.

Background & Aims Activating transcription factor 4 (ATF4) regulates genes involved in the inflammatory response, amino acid metabolism, autophagy, and endoplasmic reticulum stress. We investigated whether its activity is altered in patients with inflammatory bowel diseases (IBDs) and mice with enterocolitis. Methods We obtained biopsy samples during endoscopy from inflamed and/or uninflamed regions of the colon from 21 patients with active Crohn’s disease (CD), 22 patients with active ulcerative colitis (UC), and 38 control individuals without IBD and of the ileum from 19 patients with active CD and 8 individuals without IBD in China. Mice with disruption of Atf4 specifically in intestinal epithelial cells (Atf4ΔIEC mice) and Atf4-floxed mice (controls) were given dextran sodium sulfate (DSS) to induce colitis. Some mice were given injections of recombinant defensin α1 (DEFA1) and supplementation of l -alanyl-glutamine or glutamine in drinking water. Human and mouse ileal and colon tissues were analyzed by quantitative real-time polymerase chain reaction, immunoblots, and immunohistochemistry. Serum and intestinal epithelial cell (IEC) amino acids were measured by high-performance liquid chromatography–tandem mass spectrometry. Levels of ATF4 were knocked down in IEC-18 cells with small interfering RNAs. Microbiomes were analyzed in ileal feces from mice by using 16S ribosomal DNA sequencing. Results Levels of ATF4 were significantly decreased in inflamed intestinal mucosa from patients with active CD or active UC compared with those from uninflamed regions or intestinal mucosa from control individuals. ATF4 was also decreased in colonic epithelia from mice with colitis vs mice without colitis. Atf4ΔIEC mice developed spontaneous enterocolitis and colitis of greater severity than control mice after administration of DSS. Atf4ΔIEC mice had decreased serum levels of glutamine and reduced levels of antimicrobial peptides, such as Defa1, Defa4, Defa5, Camp, and Lyz1, in ileal Paneth cells. Atf4ΔIEC mice had alterations in ileal microbiomes compared with control mice; these changes were reversed by administration of glutamine. Injections of DEFA1 reduced the severity of spontaneous enteritis and DSS-induced colitis in Atf4ΔIEC mice. We found that expression of solute carrier family 1 member 5 (SLC1A5), a glutamine transporter, was directly regulated by ATF4 in cell lines. Overexpression of SLC1A5 in IEC-18 or primary IEC cells increased glutamine uptake and expression of antimicrobial peptides. Knockdown of ATF4 in IEC-18 cells increased expression of inflammatory cytokines, whereas overexpression of SLC1A5 in the knockdown cells reduced cytokine expression. Levels of SLC1A5 were decreased in inflamed intestinal mucosa of patients with CD and UC and correlated with levels of ATF4. Conclusions Levels of ATF4 are decreased in inflamed intestinal mucosa from patients with active CD or UC. In mice, ATF4 deficiency reduces glutamine uptake by intestinal epithelial cells and expression of antimicrobial peptides by decreasing transcription of Slc1a5. ATF4 might therefore be a target for the treatment of IBD.

Significance Paneth cells provide intestinal host defense against pathogens and control the healthy microbiota by secreting antimicrobial peptides. We show that the most abundant secreted Paneth cell products, human defensin HD-5 and HD-6, show a distinct susceptibility to proteolytic digestion by human duodenal fluid. While HD-5 is digested in many fragments, HD-6 is stable and still able to form nanonets. The occurring fragments of HD-5 were antimicrobially active against microorganisms. We provide proof of concept about microbiome modulating capacities in vivo, which includes an increase of Akkermansia sp. Our results indicate that fragmentation of defensins increases antimicrobial diversity and further adds to the complexity of host microbial interaction at interfaces. Fragmentation could lead to new antimicrobial peptides with possible therapeutic usage., Antimicrobial peptides, in particular α-defensins expressed by Paneth cells, control microbiota composition and play a key role in intestinal barrier function and homeostasis. Dynamic conditions in the local microenvironment, such as pH and redox potential, significantly affect the antimicrobial spectrum. In contrast to oxidized peptides, some reduced defensins exhibit increased vulnerability to proteolytic degradation. In this report, we investigated the susceptibility of Paneth-cell–specific human α-defensin 5 (HD-5) and -6 (HD-6) to intestinal proteases using natural human duodenal fluid. We systematically assessed proteolytic degradation using liquid chromatography–mass spectrometry and identified several active defensin fragments capable of impacting bacterial growth of both commensal and pathogenic origins. Of note, incubation of mucus with HD-5 resulted in 255–8,000 new antimicrobial combinations. In contrast, HD-6 remained stable with consistent preserved nanonet formation. In vivo studies demonstrated proof of concept that a HD-5 fragment shifted microbiota composition (e.g., increases of Akkermansia sp.) without decreasing diversity. Our data support the concept that secretion of host peptides results in an environmentally dependent increase of antimicrobial defense by clustering in active peptide fragments. This complex clustering mechanism dramatically increases the host’s ability to control pathogens and commensals. These findings broaden our understanding of host modulation of the microbiome as well as the complexity of human mucosal defense mechanisms, thus providing promising avenues to explore for drug development.

Human α-defensin 5 (HD5) is one of cationic antimicrobial peptides which plays a crucial role in an innate immune system in human body. HD5 shows the killing activity against a broad spectrum of pathogenic bacteria by making a pore in a bacterial membrane and penetrating into a cytosol. Nonetheless, its pore-forming mechanisms remain unclear. Thus, in this work, the constant-velocity steered molecular dynamics (SMD) simulation was used to simulate the permeation of a dimeric HD5 into a gram-negative LPS membrane model. Arginine-rich HD5 is found to strongly interact with a LPS surface. Upon arrival, arginines on HD5 interact with lipid A head groups and then drag these charged moieties down into a hydrophobic core resulting in the formation of water-filled pore. Although all arginines are found to interact with a membrane, R13 and R32 appear to play a dominant role in the HD5 adsorption on a gram-negative membrane. Furthermore, one chain of a dimeric HD5 is required for HD5 adhesion. The interactions of arginine-Lipid A head groups play a major role in adhering a cationic HD5 on a membrane surface and retarding a HD5 passage in the meantime.

Defensins are a family of cationic antimicrobial peptides active against a broad range of infectious microbes including bacteria, viruses and fungi, playing important roles as innate effectors and immune modulators in immunological control of microbial infection. Their antibacterial properties and unique mechanisms of action have garnered considerable interest in developing defensins into a novel class of natural antibiotic peptides to fend off pathogenic infection by bacteria, particularly those resistant to conventional antibiotics. However, serious pharmacological and technical obstacles, some of which are unique to defensins and others are common to peptide drugs in general, have hindered the development and clinical translation of defensins as anti-infective therapeutics. To overcome them, several technologies have been developed, aiming for improved functionality, prolonged circulation time, enhanced proteolytic stability and bioavailability, and efficient and controlled delivery and release of defensins to the site of infection. Additional challenges include the alleviation of potential toxicity of defensins and their cost-effective manufacturing. In this review, we briefly introduce defensin biology, focus on various transforming strategies and practical techniques developed for defensins and their derivatives as antibacterial therapeutics, and conclude with a summation of future challenges and possible solutions.

Staphylococcus hyicus is recognized as a leading pathogen of exudative epidermitis in modern swine industry. Antimicrobial peptides are attractive candidates for development as potential therapeutics to combat the serious threats of the resistance of S. hyicus. In this study, a series of derivatives were designed based on the NZ2114 template with the aim of obtaining peptides with more potent antimicrobial activity through changing net positive charge or hydrophobicity. Among them, a variant designated as NZL was highly expressed in Pichia pastoris (P. pastoris) with total secreted protein of 1505 mg/L in a 5-L fermenter and exhibited enhanced antimicrobial activity relative to parent peptide NZ2114. Additionally, NZL could kill over 99% of S. hyicus NCTC10350 in vitro within 8 h and in Hacat cells. The results of membrane permeabilization assay, morphological observations, peptide localization assay showed that NZL had potent activity against S. hyicus, which maybe kill S. hyicus through action on the cell wall. NZL also showed an effective therapy in a mouse peritonitis model caused by S. hyicus, superior to NZ2114 or ceftriaxone. Overall, these findings can contribute to explore a novel potential candidate against S. hyicus infections.

Defensins are highly conserved antimicrobial peptides, which ubiquitously expressed in different species. In addition to the functions in host defense, their aberrant expression have also been documented in cancerous tissue including breast cancer, lung caner and renal carcinoma etc. Whereas, roles of Defensin Alpha 5 (DEFA5) in colon cancer has not been explored. Bioinformatic analysis was used to study the expression of DEFA5 and its correlation with clinical outcomes; Western blot, qPCR, Co-immunoprecipitation, xenograft models were used to the study the molecular mechanism. Decreased expression of DEFA5 at protein level was observed in colon tissues. Colon cancer cell lines proliferation and colony formation capacity were significantly suppressed by DEFA5 overexpression. Moreover, in vivo tumor growth in nude mice was also suppressed by DEFA5 overexpression, suggesting a tumor suppressor role of DEFA5 in colon cancer. Mechanistically, DEFA5 directly binds to the subunits of PI3K complex, thus attenuates the downstream signaling transduction, leads to delayed cell growth and metastasis. Collectively, we concluded that DEFA5 showed an inhibitory effect in colon cancer cell growth and may serve as a potential tumor suppressor in colon cancer.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the newly emergent causative agent of coronavirus disease-19 (COVID-19), has resulted in more than two million deaths worldwide since it was first detected in 2019. There is a critical global need for therapeutic intervention strategies that can be deployed to safely treat COVID-19 disease and reduce associated morbidity and mortality. Increasing evidence shows that both natural and synthetic antimicrobial peptides (AMPs), also referred to as Host Defense Proteins/Peptides (HDPs), can inhibit SARS-CoV-2, paving the way for the potential clinical use of these molecules as therapeutic options. In this manuscript, we describe the potent antiviral activity exerted by brilacidin—a de novo designed synthetic small molecule that captures the biological properties of HDPs—on SARS-CoV-2 in a human lung cell line (Calu-3) and a monkey cell line (Vero). These data suggest that SARS-CoV-2 inhibition in these cell culture models is likely to be a result of the impact of brilacidin on viral entry and its disruption of viral integrity. Brilacidin demonstrated synergistic antiviral activity when combined with remdesivir. Collectively, our data demonstrate that brilacidin exerts potent inhibition of SARS-CoV-2 against different strains of the virus in cell culture.

During passage through the human gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) is exposed to membrane-damaging bile in the small intestine. We previously reported that EHEC treatment with a physiological bile salt mixture upregulates basRS, encoding a two-component system, and arnBCADTEF, encoding the aminoarabinose lipid A modification pathway (J. V. Kus, A. Gebremedhin, V. Dang, S. L. Tran, A. Serbanescu, and D. Barnett Foster, J Bacteriol 193: 4509–4515, 2011, https://doi.org/10.1128/JB.00200-11). The present study examined the effect of bile salt mix (BSM) treatment on EHEC resistance to three human gastrointestinal defense peptides—HD-5, HNP-1, and LL-37—as well as the role of basRS and arnT in the respective responses. After BSM treatment, EHEC resistance to HD-5 and HNP-1 was significantly increased in a BSM-, defensin dose-dependent manner. The resistance phenotype was dependent on both basRS and arnT. However, the BSM treatment did not alter EHEC resistance to LL-37, even when the ompT gene, encoding an LL-37 cleavage protease, was disrupted. Interestingly, enteropathogenic E. coli, a related pathogen that infects the small intestine, showed a similar BSM-induced resistance phenotype. Using a model of EHEC infection in Galleria mellonella, we found significantly lower survival rates in wax moth larvae infected with BSM-treated wild-type EHEC than in those infected with a BSM-treated basS mutant, suggesting that treatment with a physiological BSM enhances virulence through a basS-mediated pathway. The results of this investigation provide persuasive evidence that bile salts typically encountered during transit through the small intestine can serve as an environmental cue for EHEC, enhancing resistance to several key host defense peptides.

Moraxella catarrhalis is a bacterial pathogen that causes respiratory tract infections in humans. The increasing prevalence of antibiotic-resistant M. catarrhalis strains has created a demand for alternative treatment options. We therefore tested 23 insect antimicrobial peptides (AMPs) for their activity against M. catarrhalis in a human in vitro infection model with primary macrophages, and against commensal bacteria. Effects on bacterial growth were determined by colony counting and growth curve analysis. The inflammatory macrophage response was characterized by qPCR and multiplex ELISA. Eleven of the AMPs were active against M. catarrhalis. Defensin 1 from the red flour beetle Tribolium castaneum significantly inhibited bacterial growth and reduced the number of colony forming units. This AMP also showed antibacterial activity in the in vitro infection model, reducing cytokine expression and release by macrophages. Defensin 1 had no effect on the commensal bacteria Escherichia coli and Enterococcus faecalis. However, sarcotoxin 1 C from the green bottle fly Lucilia sericata was active against M. catarrhalis and E. coli, but not against E. faecalis. The ability of T. castaneum defensin 1 to inhibit M. catarrhalis but not selected commensal bacteria, and the absence of cytotoxic or inflammatory effects against human blood-derived macrophages, suggests this AMP may be suitable for development as a new therapeutic lead against antibiotic-resistant M. catarrhalis.

The secretions of certain cytokines, chemokines and growth factors are triggered by orthodontic appliances, which often affect the remodelling of periodontal tissues. Critical cumulative forces are applied by various types of orthodontic appliances to the periodontium. The secretion of such molecules is probably responsible, through molecular and cellular communications, for the optimal resorption of hard tissues in the periodontal setting, which therefore enables the coordination of multiple movements of tooth. This study assessed and compared a wide range of cytokines, cellular marker analysis and defensins present in the saliva samples of human subjected to orthodontic treatment with two different treatment modalities, i.e., conventional lingual and labial fixed orthodontic appliances. A total 40 samples of saliva were obtained, of which 20 were treated with traditional lingual appliances and 20 were treated with labial fixed appliances. After 21 days of treatment, all salivary samples were collected from the subjects. In order to analyse a broad range of soluble cytokine levels in saliva by flow cytometry, a bead-based immunoassay was performed. Cell surface markers were analysed by flow cytometry. Protein levels of saliva for defensins were quantified by ELISA. Non-significant differences were observed in the cytokine levels in the saliva except for the significant effects for CCL2, IL-17A and IL-6. Cellular markers CD45 and CD326 showed high percentage in conventional lingual samples. Defensin levels were found to be lower in conventional lingual patients. Subjects with conventional lingual appliances had significantly higher salivary protein levels of IL-1β, CCL2, IL17A, and IL-6, higher CD45+ and CD326+ cells and lower defensin levels than subjects with fixed labial appliances. The current study provided a clear basis for the development of innovative methods to aid in the improvement of various procedural treatments and orthodontic equipment of next generation.

In the food industry, food spoilage is a real issue that can lead to a significant amount of waste. Although current preservation techniques are being applied to reduce the occurrence of spoilage microorganisms, the problem persists. Food spoilage yeast are part of this dilemma, with common spoilers such as Zygosaccharomyces, Kluyveromyces, Debaryomyces and Saccharomyces frequently encountered. Antimicrobial peptides derived from plants have risen in popularity due to their ability to reduce spoilage. This study examines the potential application of a synthetic defensin peptide derived from barley endosperm. Its inhibitory effect against common spoilage yeasts, its mechanisms of action (membrane permeabilisation and overproduction of reactive oxygen species), and its stability in different conditions were characterised. The safety of the peptide was evaluated through a haemolysis and cytotoxicity assay, and no adverse effects were found. Both assays were performed to understand the effect of the peptide if it were to be consumed. Its ability to be degraded by a digestive enzyme was also examined for its safety. Finally, the peptide was successfully applied to different beverages and maintained the same inhibitory effects in apple juice as was observed in the antiyeast assays, providing further support for its application in food preservation.

Summary Sjögren's syndrome (SS) is a chronic autoimmune disease that mainly damages the salivary and lacrimal glands. Immune complex (IC) formation triggers local inflammation through IC deposition and decreased antigen function. Some ICs can leak from the lesion and into the saliva, but no salivary ICs have been reported to date. We used immune complexome analysis to comprehensively identify antigens incorporated into IC (IC-antigens) in saliva samples from patients with SS (n = 9) or with xerostomia (n = 7). Neutrophil defensin 1 (67%), small proline-rich protein 2D (67%), myeloperoxidase (44%), neutrophil elastase (44%), cathepsin G (33%), nuclear mitotic apparatus 1 (33%) and phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit gamma (33%) were identified as new IC-antigens specifically and frequently detected in the saliva of SS patients. Of these, neutrophil defensin 1, myeloperoxidase, neutrophil elastase and cathepsin G are neutrophil intracellular proteins, which suggests that repeated destruction of neutrophils due to abnormal autoimmunity may be involved in the pathogenesis of SS. We also analyzed serum samples from three SS patients. There was little overlap of IC-antigens between two of the samples (fewer than 30% of the IC-antigens in the saliva samples), suggesting that many ICs are formed locally and independently of the circulation. In addition, we found that four SS-specific salivary antigens show sequence homology with several proteins of oral microbiomes but no antigen has homology with Epstein–Barr virus proteins. The homology between some IC-antigens and oral microbiome proteins may indicate the impact of oral infection on local autoimmunity through molecular mimicry theory.

BACKGROUND AND AIMS: Vitamin D exerts a regulatory role over mucosal immunity via the vitamin D receptor (VDR). Although Paneth cells and their products are known to regulate the commensal and pathogenic microbiota, the role that VDRs in Paneth cells play in these responses is unknown. METHODS: We identified the decreased intestinal VDR significantly correlated with reduction of an inflammatory bowel disease risk gene ATG16L1 and Paneth cell lysozymes in patients with Crohn’s disease. We generated Paneth cell–specific VDR knockout (VDR(ΔPC)) mice to investigate the molecular mechanisms. RESULTS: Lysozymes in the Paneth cells were significantly decreased in the VDR(ΔPC) mice. Isolated VDR(ΔPC) Paneth cells exhibited weakened inhibition of pathogenic bacterial growth and displayed reduced autophagic responses. VDR(ΔPC) mice had significantly higher inflammation after Salmonella infections. VDR(ΔPC) mice also showed high susceptibility to small intestinal injury induced by indomethacin, a nonsteroidal anti-inflammatory drug. Co-housing of VDR(ΔPC) and VDR(lox) mice made the VDR(ΔPC) less vulnerable to dextran sulfate sodium colitis, suggesting the transmission of protective bacterial from the VDR(lox) mice. Thus, a lack of VDR in Paneth cells leads to impaired antibacterial activities and consequently increased inflammatory responses. Genetically and environmentally regulated VDRs in the Paneth cells may set the threshold for the development of chronic inflammation, as observed in inflammatory bowel diseases. CONCLUSIONS: We provide new insights into the tissue-specific functions of VDRs in maintaining Paneth cell alertness to pathogens in intestinal disorders. Targeting the VDR affects multiple downstream events within Paneth cells that inhibit intestinal inflammation and establish host defense against enteropathogens.

Made available in DSpace on 2022-04-29T08:45:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-08-01 Changes in the proteomic profile of the acquired enamel pellicle (AEP) formed for 3 min or 2 h after rinsing with a peptide containing the 15 N-terminal residues of statherin, with serines 2 and 3 phosphorylated (StatpSpS), were evaluated. Nine volunteers participated in 2 consecutive days. Each day, after professional tooth cleaning, they rinsed for 1 min with 10 mL of phosphate buffer containing 1.88 × 10-5 M StatpSpS or phosphate buffer only (control). The acquired pellicle formed on enamel after 3 min or 2 h was collected with electrode filter papers soaked in 3% citric acid. After protein extraction, samples were analyzed by quantitative shotgun label-free proteomics. In the 3-min AEP, 19 and 131 proteins were uniquely identified in the StatpSpS and control groups, respectively. Proteins typically found in the AEP were only found in the latter. Only 2 proteins (neutrophil defensins) were increased upon treatment with StatpSpS, while 65 proteins (among which are several typical AEP proteins) were decreased. In the 2-h AEP, 50 and 108 proteins were uniquely found in StatpSpS and control groups, respectively. Hemoglobin subunits and isoforms of keratin were only found in the StatpSpS group, while cystatin-C, cathepsin D, and cathepsin G, isoforms of heat shock 70 and protocadherin were exclusively found in the control group. In addition, 23 proteins were increased upon treatment with StatpSpS, among which are histatin-1, serum albumin, and isoforms of neutrophil defensin and keratin, while 77 were decreased, most of them were typical AEP proteins. In both evaluated periods, rinsing with StatpSpS profoundly changed the proteomic profile of the AEP, which might impact the protective role of this integument against carious or erosive demineralization. This study provides important insights on the dynamics of the protein composition of the AEP along time, after rinsing with a solution containing StatpSpS. Department of Biological Sciences Bauru School of Dentistry University of São Paulo Chemistry Institute Paulista State University Chemistry Institute Paulista State University

BackgroundMortality from bacterial meningitis, predominately caused by Streptococcus pneumoniae, exceeds 50% in sub-Saharan African countries with high HIV prevalence. Underlying causes of high mortality are poorly understood. We examined the host and pathogen proteome in the CSF of adults with proven pneumococcal meningitis (PM), testing if there was an association between differentially expressed proteins and outcome.Materials/MethodsCSF proteomes were analyzed by quantitative Mass-Spectrometry. Spectra were identified using the Swissprot human and TIGR4 pneumococcal protein libraries. Proteins were quantitated and analyzed against mortality. Unique proteins in PM were identified against published normal CSF proteome. Random-Forest models were used to test for protein signatures discriminating outcome. Proteins of interest were tested for their effects on growth and neutrophil opsonophagocytic killing of S. pneumoniae.ResultsCSF proteomes were available for 57 Adults with PM (median age 32 years, 60% male, 70% HIV-1 co-infected, mortality 63%). Three hundred sixty individual human and 23 pneumococcal proteins were identified. Of the human protein hits, 30% were not expressed in normal CSF, and these were strongly associated with inflammation and primarily related to neutrophil activity. No human protein signature predicted outcome. However, expression of the essential S. pneumoniae protein Elongation Factor Tu (EF-Tu) was significantly increased in CSF of non-survivors [False Discovery Rate (q) -correlated against expression of Neutrophil defensin (r 0.4 p p < 0.002), but not against complement proteins C3 or Factor H. In vitro, addition of EF-Tu protein impaired S. pneumoniae neutrophil killing in CSF.ConclusionsExcessive S. pneumoniae EF-Tu protein in CSF was associated with reduced survival in meningitis in a high HIV prevalence population. We show EF-Tu may inhibit neutrophil mediated killing of S. pneumoniae in CSF. Further mechanistic work is required to better understand how S. pneumoniae avoids essential innate immune responses during PM through production of excess EF-Tu.

Pregnancy is characterized by significant immunological changes and a cytokine profile, as well as vitamin deficiencies that can cause problems for the correct development of a fetus. Defensins are small antimicrobial peptides that are part of the innate immune system and are involved in several biological activities. Following that, this study aims to compare the levels of various cytokines and to investigate the role of defensins between pregnant women with confirmed COVID-19 infection and pregnant women without any defined risk factor. TNF-α, TGF-β, IL-2 and IL-10, β-defensins, have been evaluated by gene expression in our population. At the same time, by ELISA assay IL-6, IL-8, defensin alpha 1, defensin beta 1 and defensin beta 4 have been measured. The data obtained show that mothers affected by COVID-19 have an increase in pro-inflammatory factors (TNF-α, TGF-β, IL-2, IL-6, IL-8) compared to controls; this increase could generate a sort of “protection of the fetus” from virus attacks. Contemporarily, we have an increase in the anti-inflammatory cytokine IL-10 and an increase in AMPs, which highlights how the mother’s body is responding to the viral attack. These results allow us to hypothesize a mechanism of “trafficking” of antimicrobial peptides from the mother to the fetus that would help the fetus to protect itself from the infection in progress.

Antibiotics are widely used to treat various inflammatory bowel diseases caused by enterotoxigenic Escherichia coli (ETEC). However, continuous use of antibiotics may lead to drug resistance. In this study, we investigated the role of human β-defensin 118 (DEFB118) in regulating the ETEC-induced inflammation and intestinal injury. ETEC-challenged or non-challenged mice were treated by different concentrations of DEFB118. We show that ETEC infection significantly increased fecal score (P

There is abundant evidence that the innate immune response to influenza A virus (IAV) is highly complex and plays a key role in protection against IAV induced infection and illness. Unfortunately it also clear that aspects of innate immunity can lead to severe morbidity or mortality from IAV, including inflammatory lung injury, bacterial superinfection, and exacerbation of reactive airways disease. We review broadly the virus and host factors that result in adverse outcomes from IAV and show evidence that inflammatory responses can become damaging even apart from changes in viral replication per se, with special focus on the positive and adverse effects of neutrophils and monocytes. We then evaluate in detail the role of soluble innate inhibitors including surfactant protein D and antimicrobial peptides that have a potential dual capacity for down-regulating viral replication and also inhibiting excessive inflammatory responses and how these innate host factors could possibly be harnessed to treat IAV infection. Where appropriate we draw comparisons and contrasts the SARS-CoV viruses and IAV in an effort to point out where the extensive knowledge existing regarding severe IAV infection could help guide research into severe COVID 19 illness or vice versa.