STEM-20. THE ROLE OF HUMAN BETA DEFENSINS IN THE CLONOGENICITY OF GLIOBLASTOMA MULTIFORME

INTRODUCTION Glioblastoma (GBM) is the most common primary malignant brain tumor with a median overall survival of 12-15months. GBM aggressiveness and poor response to treatment are often attributed to a small population of stem-like cells referred to as glioma stem cells (GSCs). Human Beta-Defensins (HBDs), a family of small molecules initially thought to function as anti-microbials, have been implicated in various cancers with functions that are cancer type specific, including proinflammatory and immunosuppressive roles. GOAL: This study aimed to elucidate HBDs expression in GSCs and differentiated gioma cells (DGSs). METHODS We identified HBD2 and HBD3 in glioma and GSCs and DGSs by immunofluorescence (IF) and immunohistochemistry (IHC). We also assessed the role HBD2/3 play in GBM oncogenesis by investigating their effects on the self-renewal capacity of GSCs. RESULTS HBD2 and HBD3 are found in both bulk tumor and GSCs by IHC and IF. Expression of HBD2 and HBD3 mRNA levels are elevated in GBM patient tissue in comparison to lower grade gliomas by 16.2 & 1.9 fold (respectively; p < 0.0001). Additionally, transcriptional expression of HBD2 and HBD3 are increased by 0.68 and 1.15 fold respectively in GSCs versus autologous DGCs (p < 0.05; p< 0.005 respectively), suggesting a role for HBD 2/3 in oncogenesis. Interestingly however, HBD2 and HBD3 pretreatment of GSC cell lines showed decreased self-renewal capacity by 34.2 and 66.4% (p < 0.001), determined by the reduced number of large neurospheres ( >250mm). CONCLUSIONS Our results demonstrate the presence of HBD2/3 in GBM samples by IHC and IF with increased HBD2/3 mRNA expression in GBM samples. Interestingly DGCs contain less HBD2/3 than their GCS counterparts, and clonogenicity assays demonstrate a decrease in oncogenesis, suggesting that HBD’s role in proliferation and clonogenicity of DGCs and GSCs may be context dependent, leading to additional questions and future studies.

Inactivation of HIV-1 in Polarized Infant Tonsil Epithelial Cells by Human Beta-Defensins 2 and 3 Tagged with the Protein Transduction Domain of HIV-1 Tat

Mother-to-child transmission (MTCT) of HIV-1 may occur during pregnancy, labor, and breastfeeding; however, the molecular mechanism of MTCT of virus remains poorly understood. Infant tonsil mucosal epithelium may sequester HIV-1, serving as a transient reservoir, and may play a critical role in MTCT. Innate immune proteins human beta-defensins 2 (hBD-2) and -3 may inactivate intravesicular virions. To establish delivery of hBD-2 and -3 into vesicles containing HIV-1, we tagged hBDs with the protein transduction domain (PTD) of HIV-1 Tat, which facilitates an efficient translocation of proteins across cell membranes. Our new findings showed that hBD-2 and -3 proteins tagged with PTD efficiently penetrated polarized tonsil epithelial cells by endocytosis and direct penetration. PTD-initiated internalization of hBD-2 and -3 proteins into epithelial cells led to their subsequent penetration of multivesicular bodies (MVB) and vacuoles containing HIV-1. Furthermore, PTD played a role in the fusion of vesicles containing HIV-1 with lysosomes, where virus was inactivated. PTD-initiated internalization of hBD-2 and -3 proteins into ex vivo tonsil tissue explants reduced the spread of virus from epithelial cells to CD4+ T lymphocytes, CD68+ macrophages, and CD1c+ dendritic cells, suggesting that this approach may serve as an antiviral strategy for inactivating intraepithelial HIV-1 and reducing viral MTCT.

Buffalo sperm surface proteome profiling reveals an intricate relationship between innate immunity and reproduction

Background Low conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint that limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins. Results The raw mass spectra data searched against an in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a glycosylphosphatidylinositol (GPI) anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst other buffalo sperm surface proteins. The presence of these proteins was subsequently confirmed by RT-qPCR, immunofluorescence and in vitro fertilization (IVF) experiments. Conclusions The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted (glyco) proteins like BDs and the GPI-anchored proteins (GPI-APs). The glycosylation pattern of buffalo sperm-surface, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BDs on the sperm surface strengthens our hypothesis that the buffalo BDs (BuBDs) assist the spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD that could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its role in the regulation of male fertility.

Antimicrobial Peptides: Identification of Two Beta-Defensins in a Teleost Fish, the European Sea Bass (Dicentrarchus labrax)

Beta-defensins consist in a group of cysteine-rich antimicrobial peptides (AMPs), widely found throughout vertebrate species, including teleost fish, with antimicrobial and immunomodulatory activities. However, although the European sea bass (Dicentrarchus labrax) is one of the most commercially important farmed fish species in the Mediterranean area, the characterization of its beta-defensins and its potential applications are still missing. In this study, we characterized two members of the beta-defensin family in this species. Phylogenetic and synteny analysis places sea bass peptides in the beta-defensin subfamilies 1 and 2, sharing similar features with the other members, including the six cysteines and the tertiary structure, that consists in three antiparallel beta-sheets, with beta-defensin 1 presenting an extra alpha-helix at the N-terminal. Further studies are necessary to uncover the functions of sea bass beta-defensins, particularly their antimicrobial and immunomodulatory properties, in order to develop novel prophylactic or therapeutic compounds to be used in aquaculture production.

Plasma elafin, cathelicidin, and α-defensins are increased in paediatric inflammatory Crohn’s disease and reflect disease location

IntroductionThe aim of our study was to assess antimicrobial peptides in children with Crohn’s disease (CD).Material and methodsPlasma elafin, cathelicidin, alpha and beta-defensins were assessed in 35 children with CD using immunoassays. Phenotype and location of CD were assessed based on the results of endoscopic and radiology studies.ResultsWe found increased elafin, cathelicidin and alpha-defensins in children with inflammatory phenotype as compared to stricturising and penetrating phenotypes of CD. Additionally, we found increased elafin and cathelicidin in colonic location and alpha defensins in ileal CD locations.ConclusionsAssessing antimicrobial peptides may be helpful in estimating of phenotype and location of CD lesions.

The role of NOD-like receptors of innate immune cells in the pathogenesis of chronic periodontitis. Literature review

In the article, based on the data from literary sources, the features of the development of inflammato-ry periodontal diseases and immune reactions in periodontitis are considered. It is shown that the structure of innate human immunity is based on the cellular system for the rapid recognition of pathogenic microbes as for-eign bodies and their destruction. Effective recognition of pathogens BY toll-and NOD-receptors of innate im-munity causes the production of cytokines and chemokines, α-and β-beta-defensins, has an antimicrobial effect and attracts a large number of cells of the immune system to the focus of attack of microorganisms. It is concluded that the study of the exact mechanism of action of NOD receptors and their interaction with other image-recognizing receptors will lead to the development of more effective and subtle methods of controlling the in-flammatory response in periodontitis.

Buffalo Sperm Surface Proteome Profiling Reveals an Intricate Relationship Between Innate Immunity and Reproduction

BackgroundLow conception rate (CR) despite insemination with morphologically normal spermatozoa is a common reproductive restraint which limits buffalo productivity. This accounts for a significant loss to the farmers and the dairy industry, especially in agriculture-based economies. The immune-related proteins on the sperm surface are known to regulate fertility by assisting the spermatozoa in their survival and performance in the female reproductive tract (FRT). Regardless of their importance, very few studies have specifically catalogued the buffalo sperm surface proteome. The study was designed to determine the identity of sperm surface proteins and to ascertain if the epididymal expressed beta-defensins (BDs), implicated in male fertility, are translated and applied onto buffalo sperm surface along with other immune-related proteins. ResultsThe raw mass spectra data searched against in-house generated proteome database from UniProt using Comet search engine identified more than 300 proteins on the ejaculated buffalo sperm surface which were bound either by non-covalent (ionic) interactions or by a GPI-anchor. The singular enrichment analysis (SEA) revealed that most of these proteins were extracellular with varied binding activities and were involved in either immune or reproductive processes. Flow cytometry using six FITC-labelled lectins confirmed the prediction of glycosylation of these proteins. Several beta-defensins (BDs), the anti-microbial peptides including the BuBD-129 and 126 were also identified amongst the buffalo sperm surface proteins. The presence of these proteins was confirmed by immunocytochemistry and IVF experiments. Conclusions The surface of the buffalo spermatozoa is heavily glycosylated because of the epididymal secreted glycoproteins like BDs and the GPI-anchored proteins. The glycosylation pattern, however, could be perturbed in the presence of elevated salt concentration or incubation with PI-PLC. The identification of numerous BuBDs on the sperm surface strengthens our hypothesis that these BDs assist the buffalo spermatozoa either in their survival or in performance in the FRT. Our results suggest that BuBD-129 is a sperm-surface BD which could have a role in buffalo sperm function. Further studies elucidating its exact physiological function are required to better understand its roles in the regulation of male fertility.

Dysregulation of human beta-defensin-3 expression in the peripheral blood of patients with sepsis

Objectives: Sepsis is a serious medical condition caused by the body’s systemic inflammatory response to infections. The antimicrobial peptides, human beta-defensins, play a key role in modulating host immune responses, and aberrant expression of human beta-defensins has been implicated in many infections and inflammatory diseases. However, little is known about the expression of human beta-defensin-3 in systemic infectious diseases. Methods: We investigated the gene expression and protein level of human beta-defensin-3 in peripheral whole blood from 107 participants—67 patients with sepsis and 40 healthy controls—and evaluated the feasibility of human beta-defensin-3 as an indicator for sepsis. Total RNA was extracted from peripheral blood samples, and relative mRNA expression of human beta-defensin-3 was determined by reverse transcription-quantitative polymerase chain reaction. Plasma concentration of human beta-defensin-3 was measured by enzyme-linked immunosorbent assay. Pearson’s correlation analysis was performed to assess the relationship between human beta-defensin-3 mRNA and protein levels. Receiver operating characteristic analysis was performed to evaluate the value of human beta-defensin-3 as a biomarker for sepsis. Results: Human beta-defensin-3 mRNA expression was significantly downregulated in sepsis patients compared to controls (p = 0.001). The mean fold change of mRNA expression (±standard error) was 0.82 ± 0.63 in sepsis patients and 1.39 ± 1.09 in controls. Plasma concentration of human beta-defensin-3 (pg/mL) was significantly lower in sepsis patients compared to healthy controls (p = 0.039). The mean protein concentration (±standard error) was 539.6 ± 39.4 in sepsis patients and 715.5 ± 53 in controls. There was a significant correlation between human beta-defensin-3 mRNA expression and the corresponding protein level in sepsis patients (r = 0.358, p = 0.04), but not in healthy controls (r = 0.124, p = 0.51). For discriminating sepsis patients from healthy controls, the area under the receiver operating characteristic curve was 0.722 (95% confidence interval: 0.597–0.847, p = 0.002) for human beta-defensin-3 mRNA and 0.689 (95% confidence interval: 0.557–0.827, p = 0.009) for human beta-defensin-3 protein. Conclusion: This is the first study to show the downregulation of human beta-defensin-3 gene expression and protein level in sepsis, which may contribute to the complex immunological imbalance in sepsis. The significant correlation between human beta-defensin-3 mRNA expression and protein concentration suggests that mRNA expression could be used to predict protein level. Our study also showed a potential role of human beta-defensin-3 as a blood-based biomarker for sepsis. More studies on the clinical significance of human beta-defensin-3 in sepsis could further support a biomarker development.