Rabbit Red Blood Cells Research Articles

Humoral Immunity in chicken lines developed by Embrapa Suínos e Aves: Natural and specific antibodies

The antibodies produced in the first days of vertebrate life and are called natural antibodies (NAb). Other antibodies, produced in response to restricted contact with the antigen, are called specific antibodies (SpAb). To evaluate the production of NAb studies have used rabbit red blood cells (RRBC). On the other hand, evaluation of the production of specific antibodies can be performed with the use of sheep red blood cells (SRBC), a cell type that results in high production of SpAb. The aim of this study was to evaluate the production of NAb and SpAb in chicken lines developed by EMBRAPA Suínos e Aves. Animals of laying and poultry lines were inoculated intramuscularly with 5% of SRBC. The titers of NAb and SpAb were higher in laying hens than in broiler lines. The same results were obtained with anti-RRBC, the exception was males of the LLc lines, with lower antibody production compared to the other lines and females of the same line. These data show that the production of natural and specific antibodies is higher in laying hens compared to broilers, and that there is an effect of sex on antibody production.

Identification, Synthesis, and In Vitro Activities of Antimicrobial Peptide from African Catfish against the Extended-Spectrum Beta-Lactamase (ESBL)-Producing Escherichia coli.

The global surge in multi-drug resistant bacteria, including extended-spectrum β-lactamase (ESBL)-producing Escherichia coli has led to a growing need for new antibacterial compounds. Despite being promising, the potential of fish-derived antimicrobial peptides (AMPs) in combating ESBL-producing E. coli is largely unexplored. In this study, native African catfish antimicrobial peptides (NACAPs) were extracted from the skin mucus of farmed African catfish, Clarias gariepinus, using a combination of 10% acetic acid solvent hydrolysis, 5 kDa ultrafiltration, and C18 hydrophobic interaction chromatography. Peptides were then sequenced using Orbitrap Fusion Lumos Tribrid Mass Spectrometry. The identified peptides were screened for potential antibacterial activity using Random Forest and AdaBoost machine learning algorithms. The most promising peptide was chemically synthesized and evaluated in vitro for safety on rabbit red blood cells and activity against ESBL-producing E. coli (ATCC 35218) utilizing spot-on-lawn and broth dilution methods. Eight peptides ranging from 13 to 22 amino acids with molecular weights between 968.42 and 2434.11 Da were identified. Peptide NACAP-II was non-hemolytic to rabbit erythrocytes (p > 0.05) with a zone of inhibition (ZOI) of 22.7 ± 0.9 mm and a minimum inhibitory concentration (MIC) of 91.3 ± 1.2 μg/mL. The peptide is thus a candidate antibacterial compound with enormous potential applications in the pharmaceutical industry. However, further studies are still required to establish an upscale production strategy and optimize its activity and safety in vivo.

Screening the antimicrobial, cytotoxic and hemolytic effects of skin-parotoid gland secretions of 13 taxa from six species and one hybrid population of Anatolian endemic Lycian salamanders (Genus: Lyciasalamandra)

Background and purpose: The diverse bioactive content in the skin and parotoid glands of amphibians makes them unique and highly potential sources for pharmacological developments. The main aim of the study is to determine the total protein amounts, in vitro cytotoxicities, antimicrobial activities and hemolytic effects of skin-parotoid secretions of these endemic Lycian salamanders (Genus: Lyciasalamandra). Materials and methods: Lycian salamander specimens of 13 taxa from six species and one hybrid population are collected from southwestern Anatolia. Skin-parotoid secretions were obtained, clarified, supernatants snapfrozen then lyophilized. Total protein amounts were determined by BCA assay kit. The cytotoxicity was determined on eight different cell lines (one healthy and seven cancerous) using MTT assay. The antimicrobial activity was assesed using 11 different microorganisms (nine bacteria and two yeast) from six genus by MIC method. Hemolytic effects were measured on rabbit red blood cells. Results: Lycian salamanders’ skin-parotoid gland secretions showed variable cytotoxic effects on all cell lines with IC50 values from 5.41 ± 0.17 μg/ml to ineffective. MIC results of antimicrobial activity tests were also variable and from 3.9 μg/mL to non-effective. Studied skin-parotoid secretions have no hemolytic activities on rabbit red blood cells at concentrations of 0.5, 5 and 50 μg/mL. Conclusions: Skin-parotoid gland secretions of Anatolian endemic Lycian salamanders have sufficient and remarkable therapeutic potential to be candidate sources of natural and biologically active substances for novel drug discoveries.

Construction of GPC3-modified Lipopolymer SiRNA Delivery System.

Gene therapy has been widely concerned because of its unique therapeutic mechanism. However, due to the lack of safe and effective carries, it has not been widely used in clinical practice. Glypican 3 (GPC3) is a highly specific proteoglycan for hepatocellular carcinoma and is a potential diagnostic and therapeutic target for hepatocellular carcinoma. Herein, to monitor the effect of gene therapy and enhance the transfection efficiency of gene carriers, GPC3-modified lipid polyethyleneimine-modified superparamagnetic nanoparticle (GLPS), a type of visualized carrier for siRNA (small-interfering RNA) targeting the liver, was prepared. We performed in vitro gene silencing, cytotoxicity, and agarose gel electrophoresis to identify the optimal GLPS formulation. In vitro MRI and Prussian blue staining verified the liver-targeting function of GLPS. We also analyzed the biocompatibility of GLPS by co-culturing with rabbit red blood cells. Morphological changes were evaluated using HE staining. The GLPS optimal formulation consisted of LPS and siRNA at a mass ratio of 25:1 and LPS and DSPE-PEG-GPC3 at a molar ratio of 2:3. GLPS exhibited evident liver-targeting function. In vitro, we did not observe morphological changes in red blood cells or hemolysis after co-culture. In vivo, routine blood analysis revealed no abnormalities after GLPS injection. Moreover, the tissue morphology of the kidney, spleen, and liver was normal without injury or inflammation. GLPS could potentially serve as an effective carrier for liver-targeted MRI monitoring and siRNA delivery.

Genome-wide characterization, phylogenetic and expression analysis of Galectin gene family in Golden pompano Trachinotus ovatus.

Galectins (Gals) are a type of S-type lectin that are widespread and evolutionarily conserved among metazoans, and can act as pattern recognition receptors (PRRs) to recognize pathogen-associated molecular patterns (PAMPs). In this study, 10 Gals (ToGals) were identified in the Golden pompano (Trachinotus ovatus), and their conserved domains, motifs, and collinearity relationships were analyzed. The expression of ToGals was regulated following infection to Cryptocaryon irritans and Streptococcus agalactiae, indicating that ToGals participate in immune responses against microbial pathogens. Further analysis was conducted on one important member, Galectin-3, subcellular localization showing that ToGal-3like protein is expressed both in the nucleus and cytoplasm. Recombinant protein obtained through prokaryotic expression showed that rToGal-3like can agglutinate red blood cells of rabbit, carp and golden pompano and also agglutinate and kill Staphylococcus aureus, Bacillus subtilis, Vibrio vulnificus, S. agalactiae, Pseudomonas aeruginosa, and Aeromonas hydrophila. This study lays the foundation for further research on the immune roles of Gals in teleosts.

Identification and characterization of β3 integrin in Crassostrea hongkongensis

Crassostrea hongkongensis is a main oyster species farmed in South China Sea offshore area, and commonly suffers from vibriosis. Vibrio parahaemolyticus is one of the principal pathogens that causes the oyster vibriosis. The integrin from C. hongkongensis was studied for its function against V. parahaemolyticus in the current work. The full-length cDNA sequence was 3274 bp with an open reading frame (ORF) of 2397 bp that encoded a polypeptide of 798 amino acids. The polypeptide included typical domains of β integrin (such as INB domain, three EGF-like domains, and highly conserved NPX[Y/F] motif), and phylogenetically clustered with β3 integrins of other invertebrates, thereby, designated as ChIntβ3. The mRNA transcripts of ChIntβ3 were significantly higher in the heart, hemocytes, adductor muscle and hepatopancreas than those in gills and mantle of C. hongkongensis. Following stimulation with V. parahaemolyticus, the mRNA expression level of ChIntβ3 was significantly up-regulated in both hemocytes and hepatopancreas. The recombinant ChIntβ3 had significantly agglutinating activity on Escherichia coli, V. parahaemolyticus, Bacillus amyloliquefaciens, Pichia pastoris and the red blood cells of rabbit. In the one hand, the survival rate (83.33 ± 5.77%) of the oyster after V. parahaemolyticus infection at 30 days was significantly higher in the ChIntβ3 overexpression group than those in the physiological saline control group (26.67 ± 20.82%) (p < 0.01) and the overexpressed GFP group (26.67 ± 5.77%) (p < 0.01); in the other hand, the cumulative survival duration of C. hongkongensis was significantly shorter in the RNAi treatment group (197.6 ± 2.77 h) than that in the control group (247.2 ± 36.24 h) (p < 0.01). The results indicated that ChIntβ3 was involved in the recognition and clearance of V. parahaemolyticus through cell adhesion, and contributed to the further understanding of the function of ChIntβ3 against infection of pathogens.

Complement dysregulation associated with a genetic variant in factor H-related protein 5 in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) can be associated with mutations, deletions, or hybrid genes in factor H-related (FHR) proteins. A child with aHUS was investigated. Genetics was assessed by Sanger and next generation sequencing. Serum FHR5 was evaluated by immunoblotting, ELISA, and by induction of rabbit red blood cell hemolysis in the presence/absence of recombinant human rFHR5. Mutagenesis was performed in HEK cells. A heterozygous genetic variant in factor H-related protein 5 (CFHR5), M514R, was found in the child, who also had a homozygous deletion of CFHR3/CFHR1, and antibodies to factor H, as well as low levels of C3. Patient serum exhibited low levels of FHR5. In the presence of rabbit red blood cells, patient serum induced hemolysis which decreased when rFHR5 was added at physiological concentrations. Similar results were obtained using serum from the father, bearing the CFHR5 variant without factor H antibodies. Patient FHR5 formed normal dimers. The CFHR5 M514R variant was expressed in HEK cells and minimal secretion was detected whereas the protein level was elevated in cell lysates. Decreased secretion of the product of the mutant allele could explain the low FHR5 levels in patient serum. Reduced hemolysis when rFHR5 was added to serum suggests a regulatory role regarding complement activation on red blood cells. As such, low levels of FHR5, as demonstrated in the patient, may contribute to complement activation.

Molecular cloning, expression, and functional analysis of a putative lectin from the pearl oyster (Pinctada fucata, Gould 1850)

Marine lectins are a group of proteins that possess specific carbohydrate recognition and binding domains. They exhibit various activities, including antimicrobial, antitumor, antiviral, and immunomodulatory effects. In this study, a novel galectin-binding lectin gene named PFL-96 (GenBank: OQ561753.1) was cloned from Pinctada fucata. The PFL-96 gene has an open reading frame of 324 base pairs (bp) and encodes a protein comprising 107 amino acids. The protein has a molecular weight of 11.95 kDa and an isoelectric point of 9.27. It contains an N-terminal signal peptide and a galactose-binding lectin domain. The sequence identity to lectin proteins from fish, echinoderms, coelenterates, and shellfish ranges from 31.90 to 40.00 %. In the phylogenetic analysis, it was found that the PFL-96 protein is closely related to the lectin from Pteria penguin. The PFL-96 recombinant protein exhibited coagulation activity on 2 % rabbit red blood cells at a concentration of ≥8 μg/mL. Additionally, it showed significant hemolytic activity at a concentration of ≥32 μg/mL. The PFL-96 recombinant protein exhibited significant antibacterial activity against Bacillus subtilis, Staphylococcus aureus, Candida albicans, and Vibrio alginolyticus, with minimum inhibitory concentrations (MIC) of 4, 8, 16, and 16 μg/mL, respectively. The minimum bactericidal concentrations (MBC) were determined to be 8, 16, 32, and 32 μg/mL, respectively. Furthermore, the PFL-96 recombinant protein exhibited inhibitory effects on the proliferation of Hela tumor cells, HepG2 tumor cells, and C666-1 tumor cells, with IC50 values of 7.962, 8.007, and 9.502 μg/mL, respectively. These findings suggest that the recombinant protein PFL-96 exhibits significant bioactivity in vitro, contributing to a better understanding of the active compounds found in P. fucata. The present study establishes a fundamental basis for further investigation into the mechanism of action and structural optimization of the recombinant protein PFL-96. The aim is to develop potential candidates for antibacterial and anti-tumor agents.

Loaded delta-hemolysin shapes the properties of Staphylococcus aureus membrane vesicles.

Membrane vesicles (MVs) are nanoscale vesicular structures produced by bacteria during their growth in vitro and in vivo. Some bacterial components can be loaded in bacterial MVs, but the roles of the loaded MV molecules are unclear. MVs of Staphylococcus aureus RN4220 and its derivatives were prepared. Dynamic light scattering analysis was used to evaluate the size distribution, and 4D-label-free liquid chromatography-tandem mass spectrometry analysis was performed to detect protein composition in the MVs. The site-mutation S. aureus RN4220-Δhld and agrA deletion mutant RN4220-ΔagrA were generated via allelic replacement strategies. A hemolysis assay was performed with rabbit red blood cells. CCK-8 and lactate dehydrogenase release assays were used to determine the cytotoxicity of S. aureus MVs against RAW264.7 macrophages. The serum levels of inflammatory factors such as IL-6, IL-1β, and TNFα in mice treated with S. aureus MVs were detected with an enzyme-linked immunosorbent assay kit. Delta-hemolysin (Hld) was identified as a major loaded factor in S. aureus MVs. Further study showed that Hld could promote the production of staphylococcal MVs with smaller sizes. Loaded Hld affected the diversity of loaded proteins in MVs of S. aureus RN4220. Hld resulted in decreased protein diversity in MVs of S. aureus. Site-mutation (RN4220-Δhld) and agrA deletion (RN4220-ΔagrA) mutants produced MVs (ΔhldMVs and ΔagrAMVs) with a greater number of bacterial proteins than those derived from wild-type RN4220 (wtMVs). Moreover, Hld contributed to the hemolytic activity of wtMVs. Hld-loaded wtMVs were cytotoxic to macrophage RAW264.7 cells and could stimulate the production of inflammatory factor IL-6 in vivo. This study presented that Hld was a major loaded factor in S. aureus MVs, and the loaded Hld played vital roles in the MV-property modification.

Enhanced wound healing and hemostasis with exosome-loaded gelatin sponges from human umbilical cord mesenchymal stem cells.

Rapid wound healing remains a pressing clinical challenge, necessitating studies to hasten this process. A promising approach involves the utilization of human umbilical cord mesenchymal stem cells (hUC-MSCs) derived exosomes. The hypothesis of this study was that these exosomes, when loaded onto a gelatin sponge, a common hemostatic material, would enhance hemostasis and accelerate wound healing. To investigate the hemostatic and wound healing efficacy of gelatin sponges loaded with hUC-MSCs-derived exosomes. Ultracentrifugation was used to extract exosomes from hUC-MSCs. Nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM), and western blot techniques were used to validate the exosomes. In vitro experiments were performed using L929 cells to evaluate the cytotoxicity of the exosomes and their impact on cell growth and survival. New Zealand rabbits were used for skin irritation experiments to assess whether they caused adverse skin reactions. Hemolysis test was conducted using a 2% rabbit red blood cell suspension to detect whether they caused hemolysis. Moreover, in vivo experiments were carried out by implanting a gelatin sponge loaded with exosomes subcutaneously in Sprague-Dawley (SD) rats to perform biocompatibility tests. In addition, coagulation index test was conducted to evaluate their impact on blood coagulation. Meanwhile, SD rat liver defect hemostasis model and full-thickness skin defect model were used to study whether the gelatin sponge loaded with exosomes effectively stopped bleeding and promoted wound healing. The NTA, TEM, and western blot experimental results confirmed that exosomes were successfully isolated from hUC-MSCs. The gelatin sponge loaded with exosomes did not exhibit significant cell toxicity, skin irritation, or hemolysis, and they demonstrated good compatibility in SD rats. Additionally, the effectiveness of the gelatin sponge loaded with exosomes in hemostasis and wound healing was validated. The results of the coagulation index experiment indicated that the gelatin sponge loaded with exosomes had significantly better coagulation effect compared to the regular gelatin sponge, and they showed excellent hemostatic performance in a liver defect hemostasis model. Finally, the full-thickness skin defect healing experiment results showed significant improvement in the healing process of wounds treated with the gelatin sponge loaded with exosomes compared to other groups. Collectively, the gelatin sponge loaded with hUC-MSCs-derived exosomes is safe and efficacious for promoting hemostasis and accelerating wound healing, warranting further clinical application.

Pattern recognition receptors involved in the immune system of hagfish (Eptatretus burgeri)

The initial defense against invading pathogenic microbes is the activation of innate immunity by binding of pattern recognition receptors (PRRs) to pathogen associated molecular patterns (PAMPs). To explain the action of PRRs from hagfish, one of the extant jawless vertebrates, we purified the GlcNAc recognition complex (GRC) from serum using GlcNAc-agarose. The GRC comprises four proteins of varying molecular masses: 19 kDa, 26 kDa, 27 kDa, and 31 kDa. Exposure of Escherichia coli to the GRC led to the phagocytic activation of macrophages, revealing the opsonic function of the GRC. The GRC in serum formed a large complex with a molecular mass of approximately 1200 kDa. The GRC bound to Escherichia coli but not to rabbit red blood cells, despite both having GlcNAc on their surface. These structural and binding properties are similar to those of mannose-binding lectin (MBL). The amino acid sequence of a portion of the 31 kDa protein in the GRC matched the amino acid sequence of variable lymphocyte receptor (VLR)-B in some place. According to the Western blot analysis, the 31 kDa protein was recognized by the anti-hagfish VLR-B antiserum. Based on the results, it appears that the GRC functions as a PRR like MBL and that its 31 kDa protein has a structure similar to that of VLR-B.

Metal-ruthenium complex based on dipyridylamine group as membrane-active antibacterial agent effectively decrease the development of drug-resistance on Staphylococcus aureus

Staphylococcus aureus (S. aureus), one of the Gram-positive bacteria, is easily to develop drug-resistance. Drug-resistant S. aureus infection leads to high morbidity and mortality. The complexes, namely [Ru(dpa)2(PSPIP)](PF6)2 (Ru1), [Ru(dpa)2(TSPIP)](PF6)2 (Ru2), and [Ru(dpa)2(TBPIP)](PF6)2 (Ru3), were synthesized using 2, 2′-dipyridylamine as an auxiliary ligand and three main ligands PSPIP, TSPIP, TBPIP. In vitro studies demonstrated that the Ru1–3 exhibited excellent antibacterial activity against S. aureus while showing low hemolytic toxicity to rabbit red blood cells. Notably, Ru3 was found to disrupt the bacterial cell membrane and alter its permeability through fluorescence staining and scanning electron microscopy (SEM) analysis. Furthermore, Ru3 displayed low toxicity in G. mellonella Larvae. Ru3 exhibited good activity against S. aureus in G. mellonella Larvae infection model and mouse skin infection model.To some extent, Ru3 inhibited biofilm formation on S. aureus as well as hemolytic toxin production, thereby attenuating the development of drug resistance without cross-resistance with other antibiotics. In addition, complex Ru3 exhibited a synergistic effect when combined with antibiotics amikacin, kanamycin, tobramycin and chloramphenicol, making it a valuable antibiotics adjuvant.

Purification and characterization of an asialofetuin specific lectin from the rhizome of Xanthosoma violaceum Schott

Lectins are proteins or glycoproteins that bind specifically and reversibly to the carbohydrate or glycoconjugates. A new lectin is purified from the rhizome of Xanthosoma violaceum Schott. by successive steps of ammonium sulfate fractionation and affinity chromatography with asialofetuin as ligand. The purified lectin was found to be a homotetramer of approximately 49 kDa with a subunit molecular weight of 12 kDa linked by non-covalent bonds. Characterization of the lectin shows that the hemagglutination activity is inhibited by asialofetuin and d-galacturonic acid. Hemagglutination activity is shown only in rabbit RBC but not in the human RBC of all blood groups. It is a metal ion-independent glycoprotein of 1.87% carbohydrate content, stable upto 40 °C and pH from 5.5 to 9. The lectin shows its optimum hemagglutination activity at 0 °C–40 °C and pH 6 to 8.5. From LC-MS/MS analysis it is confirmed that the purified lectin was not purified and characterized earlier.

The effects of the differentiated macrophages by dexamethasone on the immune responses

The characteristics of M2 macrophages suggest immunotherapeutic approaches for inducing immunological tolerance. The current study aimed to evaluate the effect of Dexamethasone (Dex) treatment on monocytes polarization and its impact on immune responses. The monocytes were extracted from the rat's blood samples. The effects of Dex concentration and treatment duration on monocyte viability, phagocytosis of rabbit red blood cell (RRBC) antigens, and cytokine gene expression were evaluated using MTT, ELISA, and Real-Time PCR analysis, respectively. The monocytes treated with Dex were injected into the rats as an autograft. The effects of the grafted cells were assessed on immune responses, monocyte differentiation, and pathological lesions, in comparison to the control groups.Treatment of monocytes with 10-5 M of Dex for 48 h increased the expression of IL-10 and TGF-β genes, while reducing the expression of TNF-α and IL-1 genes. The monocytes treated with antigen and Dex showed higher CD206 gene expression compared to CD80. The cells that were treated with Dex had the highest concentration of antigens after five days. Administration of the grafted cells to the animals has some significant effects on innate immune responses and no impact on pathological lesions. The group that received cells treated with Dex and antigen experienced a significant decrease in anti-RBC antibody titers. Additionally, there was a significant difference in the expression of cytokine genes and M2 differentiation markers among the groups that were evaluated. The effects of Dex on the viability and differentiation of monocytes depend on the dosage, timing, and duration of the treatment.

An automated reading of semi-quantitative hemagglutination results in microplates: Micro-assay for plant lectins

Abstract An automated, qualitative and semi-quantitative micro-assay for the detection of plant lectins also known as phyto-agglutinins (glycoproteins; have exciting applications in medicines) is described as an alternative to conventional assays. The method developed in this work is based on hemagglutination (HA) assay that can simultaneously detect the presence and concentration (titer) of lectins in as many as 96 different samples without the aid of an expert eye. We used rice (Oryza sativa L.) seeds for making clarified lectin extracts and Arabic gum as positive control in phosphate buffer saline; the method is applicable to all kinds and parts of the plants. Rabbit red blood cells were used in order to carry out the HA assay in a miniaturized experiment using U-welled microplates (MPs). 25 µL of plant extract is sufficient to carry out HA micro-assay at incubation temperature of 38°C for 20 min. The method was standardized with an expert eye and automated by using MP reader. Moreover, a standard curve for the direct interpretation of lectin concentration is also developed by conversion of absorbance values into titer. The method described will save time, material, labor, and simplify the rapid semi-quantification of plant lectins.

MASP3 Deficiency in Mice Reduces but Does Not Abrogate Alternative Pathway Complement Activity Due to Intrinsic Profactor D Activity.

Complement factor D (FD) is a rate-limiting enzyme of the alternative pathway (AP). Recent studies have suggested that it is synthesized as an inactive precursor and that its conversion to enzymatically active FD is catalyzed by mannan-binding lectin-associated serine protease 3 (MASP3). However, whether MASP3 is essential for AP complement activity remains uncertain. It has been shown that Masp1/3 gene knockout did not prevent AP complement overactivation in a factor H-knockout mouse, and a human patient lacking MASP3 still retained AP complement activity. In this study, we have assessed AP complement activity in a Masp3-knockout mouse generated by CRISPR/Cas9 editing of the Masp1/3 gene. We confirmed specific Masp3 gene inactivation by showing intact MASP1 protein expression and absence of mature FD in the mutant mice. Using several assays, including LPS- and zymosan-induced C3b deposition and rabbit RBC lysis tests, we detected plasma concentration-dependent AP complement activity in Masp3 gene-inactivated mice. Thus, although not measurable in 5% plasma, significant AP complement activity was detected in 20-50% plasma of Masp3 gene-inactivated mice. Furthermore, whereas FD gene deletion provided more than 90% protection of CD55/Crry-deficient RBCs from AP complement-mediated extravascular hemolysis, Masp3 gene deletion only provided 30% protection in the same study. We also found pro-FD to possess intrinsic catalytic activity, albeit at a much lower level than mature FD. Our data suggest that MASP3 deficiency reduces but does not abrogate AP complement activity and that this is explained by intrinsic pro-FD activity, which can be physiologically relevant invivo.

Characteristics of antimicrobial peptide OaBac5mini and its bactericidal mechanism against Escherichia coli.

Antimicrobial peptides (AMPs) play an important role in defending against the attack of pathogenic microorganisms. Among them, the proline-rich antibacterial peptides (PrAMPs) have been attracting close attention due to their simple structure, strong antibacterial activity, and low cell toxicity. OaBac5mini is an active fragment of the sheep-derived OaBac5 belonging to the PrAMPs family. In this study, the antibacterial activity of OaBac5mini was investigated by testing the MICs against different stains of E. coli and S. aureus as well as the time-kill curve. The bactericidal mechanism was explored by determining the effect of OaBac5mini on the cell membrane. The stability and biosafety were also evaluated. The susceptibility test demonstrated that OaBac5mini showed potent antibacterial activity against the multidrug-resistant (MDR) E. coli isolates. It is noticeable that the absence of inner membrane protein SbmA in E. coli ATCC 25922 caused the MIC of OaBac5mini to increase 4-fold, implying OaBac5mini can enter into the cytoplasm via SbmA and plays its antibacterial activity. Moreover, the antibacterial activity of OaBac5mini against E. coli ATCC 25922 was not remarkably affected by the serum salts except for CaCl2 at a physiological concentration, pH, temperature, repeated freeze-thawing and proteases (trypsin < 20 μg/mL, pepsin or proteinase K). Time-kill curve analysis showed OaBac5mini at the concentration of 200 μg/mL (8 × MICs) could effectively kill E. coli ATCC 25922 after co-incubation for 12 h. In addition, OaBac5mini was not hemolytic against rabbit red blood cells and also was not cytotoxic to porcine small intestinal epithelial cells (IPEC-J2). Bioinformatic analysis indicated that OaBac5mini is a linear peptide with 8 net positive charges. Furthermore, OaBac5mini significantly increased the outer membrane permeability and impaired the inner membrane integrity and ultrastructure of E. coli ATCC25922. OaBac5mini is a stable and potent PrAMP that kills E. coli by two different modes of action - inhibiting intracellular target(s) and damaging cell membrane.

Autoantibodies against complement factor B in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder affecting the joints. Many patients carry anti-citrullinated protein autoantibodies (ACPA). Overactivation of the complement system seems to be part of the pathogenesis of RA, and autoantibodies against the pathway initiators C1q and MBL, and the regulator of the complement alternative pathway, factor H (FH), were previously reported. Our aim was to analyze the presence and role of autoantibodies against complement proteins in a Hungarian RA cohort. To this end, serum samples of 97 ACPA-positive RA patients and 117 healthy controls were analyzed for autoantibodies against FH, factor B (FB), C3b, C3-convertase (C3bBbP), C1q, MBL and factor I. In this cohort, we did not detect any patient with FH autoantibodies but detected C1q autoantibodies in four patients, MBL autoantibodies in two patients and FB autoantibodies in five patients. Since the latter autoantibodies were previously reported in patients with kidney diseases but not in RA, we set out to further characterize such FB autoantibodies. The isotypes of the analyzed autoantibodies were IgG2, IgG3, IgGκ, IgGλ and their binding site was localized in the Bb part of FB. We detected in vivo formed FB-autoanti-FB complexes by Western blot. The effect of the autoantibodies on the formation, activity and FH-mediated decay of the C3 convertase in solid phase convertase assays was determined. In order to investigate the effect of the autoantibodies on complement functions, hemolysis assays and fluid phase complement activation assays were performed. The autoantibodies partially inhibited the complement-mediated hemolysis of rabbit red blood cells, inhibited the activity of the solid phase C3-convertase and C3 and C5b-9 deposition on complement activating surfaces. In summary, in ACPA-positive RA patients we identified FB autoantibodies. The characterized FB autoantibodies did not enhance complement activation, rather, they had inhibitory effect on complement. These results support the involvement of the complement system in the pathomechanism of RA and raise the possibility that protective autoantibodies may be generated in some patients against the alternative pathway C3 convertase. However, further analyses are needed to assess the exact role of such autoantibodies.

Characterization of a Lipopolysaccharide- and Beta-1,3-Glucan Binding Protein (LGBP) from the Hepatopancreas of Freshwater Prawn, Macrobrachium rosenbergii, Possessing Lectin-Like Activity.

The study focuses on the isolation, characterization, and expression analysis of a lectin from the hepatopancreas of Macrobrachium rosenbergii. The protein was isolated by affinity chromatography on a melibiose-agarose column. The molecular weight of the native protein was found to be ~120kDa which consists of a single polypeptide of ~39.5kDa. On mass spectrometric analysis, the protein was identified as lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP). LGBP showed hemagglutination with rabbit RBC like a lectin and its carbohydrate-binding specificity was determined by the hemagglutination inhibition test. The protein also showed antibacterial activity against two Gram-negative bacteria Vibrio harveyi and Aeromonas sobria, and one Gram positive bacteria Bacillus cereus in the disc diffusion test. Rabbit antiserum was raised against the purified LGBP and used to develop a sandwich ELISA system for quantitation of the protein in hepatopancreas and serum samples of M. rosenbergii. The expression of the LGBP transcripts in muscle, hepatopancreas, and gill tissues from M. rosenbergii juveniles at 72h post-challenge of V. harveyi was not modulated as noticed in qPCR analysis. However, significant increases in the concentrations of LGBP protein in hepatopancreas (5.23 ± 0.45 against 3.43 ± 0.43mg/g tissue in control) and serum (1.08 ± 0.14 against 0.61 ± 0.08µg/ml in control) were observed in the challenged group of prawns in ELISA suggesting its putative role against bacterial infections. The study for the first time characterized the native LGBP of M. rosenbergii showing a multifunctional role in immunity.

Identification of two neutralizing human single-chain variable fragment antibodies targeting Staphylococcus aureus alpha-hemolysin.

Objective(s):The inability of the host immune system to defeat Staphylococcus aureus is due to various secreted virulent factors such as leukocidins, superantigens, and hemolysins, which interrupt the function of immune components. Alpha-hemolysin is one of the most studied cytolysins due to its pronounced effect on developing staphylococcal infections. Alpha-hemolysin-neutralizing antibodies are among the best candidates for blocking the toxin activity and preventing S. aureus pathogenesis. Materials and Methods:A human single-chain variable fragment (scFv) phage display library was biopanned against alpha-hemolysin. The selected phage clones were assessed based on their binding ability to alpha-hemolysin. The binding specificity and affinity of two scFvs (designated SP192 and SP220) to alpha-hemolysin were determined by enzyme-linked immunosorbent assay. Furthermore, the neutralizing activity of SP192 and SP220 was examined by concurrent incubation of rabbit red blood cells (RBCs) with alpha-hemolysin and scFvs.Results:SP192 and SP220 showed significant binding to alpha-hemolysin compared with the control proteins, including bovine serum albumin, human adiponectin, and toxic shock syndrome toxin-1. Besides, both scFvs showed high-affinity binding to alpha-hemolysin in the nanomolar range (Kaff: 0.9 and 0.7 nM-1, respectively), leading to marked inhibition of alpha-hemolysin-mediated lysis of rabbit RBCs (73% and 84% inhibition; respectively). Conclusion:SP192 and SP220 scFvs can potentially be used as alpha-hemolysin-neutralizing agents in conjunction with conventional antibiotics to combat S. aureus infections.