Serum interleukin-10 and alpha-fetoprotein: A combined diagnostic approach for hepatocellular carcinoma in Egyptians with HCV.

PURPOSE: Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Although alpha-fetoprotein (AFP) has been used for 60+ years as an HCC diagnostic serum marker, its accuracy is debated. Notably, the role of interleukin 10 (IL-10) in cancer development and metastasis is elevated in various tumor types, including HCC and chronic HCV infection. Our study aimed to investigate the diagnostic performance of IL-10 and AFP as biomarkers for HCV-induced HCC in an Egyptian population. METHODS: Eighty participants were recruited and categorized into three groups: HCV-related HCC (n=40), HCV-related cirrhosis (n=40), and control (n=20).The collected blood samples were analyzed to evaluate liver function, AFP levels, and IL-10 levels. RESULTS: Our analysis showed that AFP demonstrated low sensitivity (40% false-negative) and low specificity (33% false-positive).IL-10 levels were significantly higher (P < 0.001) in patients with HCC than in the cirrhosis and control groups. The serum AFP and IL-10 combination revealed significantly increased sensitivity (97.5%), diagnostic accuracy (71.1%), AUC (0.798), PPV (73.3%), and NPV ( 69.5%) when compared with either of them alone. CONCLUSION: the reliability of AFP as a major HCC marker was poor. However, IL-10 levels are a novel biomarker for the degree of HCC inflammation, considering IL-10's potential role in HCV-HCC development. We suggest combining AFP with IL-10 to improve the diagnostic and prognostic value of HCC considerably. Future research on these biomarkers should prioritize their clinical validity, prognostic usefulness, and compatibility with other therapeutic approaches as immunotherapy.

Photobiological modulation of hepatoma cell lines and hepatitis B subviral particles secretion in response to 650 nm low level laser treatment.

BACKGROUND: Chronic hepatitis B virus (HBV) infection is a serious global health concern, with an increased incidence and risk of developing cirrhosis and hepatocellular carcinoma (HCC). Patients chronically infected with HBV are likely to experience chronic oxidative stress, leading to mitochondrial dysfunction. Photobiomodulation is induced by the absorption of low-level laser therapy (LLLT) with a red or infrared laser by cytochrome C oxidase enzyme, resulting in mitochondrial photoactivation. Although it is widely used in clinical practice, the use of LLL as adjuvant therapy for persistent HBV infection is uncommon. This study aimed to investigate the effect of LLLT dosage from 2 J/cm2 to 10 J/cm2 of red diode laser (650 nm) on both hepatoma cell lines (HepG2.2.15 [integrated HBV genome stable cell model] and non-integrated HepG2), with a subsequent impact on HBVsvp production. METHODS: The present study evaluated the effects of different fluences of low-level laser therapy (LLLT) irradiation on various aspects of hepatoma cell behavior, including morphology, viability, ultrastructure, and its impact on HBVsvp synthesis. RESULTS: In response to LLLT irradiation, we observed a considerable reduction in viability, proliferation, and HBVsvp production in both hepatoma cell lines HepG2.2.15 and HepG2. Ultrastructural modification of mitochondria and nuclear membranes: This effect was dose, cell type, and time-dependent. CONCLUSIONS: The use of LLLT may be a promising therapy for HCC and HBV patients by reducing cell proliferation, HBVsvp production, and altering mitochondrial and nuclear structure involved in cellular death inducers. Further research is required to explore its clinical application.

In vitro Anti SARS-CoV-2 Activity and Docking Analysis of Pleurotus ostreatus, Lentinula edodes and Agaricus bisporus Edible Mushrooms.

Background: Fungi are rich source of biologically active metabolites aimed for the improvement of human health through the prevention of various diseases, including infections and inflammatory disorders. Aim: We aimed to in vitro examine the anti-SARS CoV-2 activity of the aqueous extract of each Pleurotus (P.) ostreatus, Lentinula (L.) edodes and Agaricus (A.) bisporus edible mushroom followed by docking analysis of certain metabolites against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-main protease (protease Mpro). Methods: Antiviral and cytotoxic effects were tested on hCoV-19/Egypt/NRC-3/2020/Vero-E6 cells and analyzed via (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide Assay (MTT) assay. Ligand-protein and protein-protein docking studies were performed to explore the interaction of different mushroom extracts at the binding site of protease Mpro. Molecular dynamics (MD) simulations were performed on the most promising ligand-target complexes to investigate their dynamic properties and confirm docking results. Results: Substantial antiviral activities with an IC50 of 39.19, 26.17, and 10.3.3 µg/mL and a selectivity index (SI) of 4.34, 3.44, and 1.5 for P. ostreatus, L. edodes and A. bisporus, were observed, respectively. Docking analysis revealed that, catechin from three mushroom isolates, chlorogenic acid from A. bisporus, kamperferol of P. ostreatus and quercetin from L. edodes, with a C-DOCKER interaction energy in the range of 22.8-37.61 (Kcal/mol) with protease compared to boceprevir ligand of 41.6 (Kcal/mol). Docking of superoxide dismutase, catalase from the three mushrooms, tyrosinase from A. bisporus showed ligand contact surface area with the protein as 252.74 Å2 while receptor contact surface area was 267.23 Å2. Conclusion: P. ostreatus, L. edodes and A. bisporus have potential and remarkable in vitro antiviral activities against SARS-CoV-2. Quercetin from L. edodes, Kaempferol from P. ostreatus, chlorogenic acid and ascorbic acid, catechin, superoxide dismutase and catalase of the three mushrooms extracts were effectively bounded to Mpro of SARS-CoV-2 as conferred by docking analysis.

Immunomodulatory activity of extracts from five edible basidiomycetes mushrooms in Wistar albino rats.

Mushrooms are nutritious foods that are widely cultivated all over the world. They are rich in a range of compounds linked to improving functions of the immune system including carotenoids, alkaloids, lectins, enzymes, folates, fats, organic acids, minerals, polysaccharides, phenolics, proteins, tocopherols, terpenoids, and volatile compounds. In this study we investigated, the immunomodulatory activity in rats of the aqueous extracts of five of the most common edible mushrooms belonging to Family Basidiomycota-white-rot fungi including, Lentinula edodes, Agaricus bisporus, Pleurotus ostreatus, Pleurotus columbinus, and Pleurotus sajor-caju. Male Wistar albino rats were assigned to thirteen groups and Immunosuppression was induced by oral administration of dexamethasone (0.1 mg/kg), followed by oral administration of the mushroom extracts at low (200 mg/kg) and high (400 mg/kg) doses. A positive control group received the immune stimulant Echinacea extract Immulant® at (30 mg/kg), while the negative control group received only saline. From each animal, in each group, blood samples were collected after 15 days for complete blood counts and for measurement of immunologic parameters, including lysozyme activity, nitric oxide (NO) production and serum cytokines including tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ) and interleukin 1 beta (IL-1ß) levels. Results have shown that white blood cells (WBCs) and lymphocytic counts were significantly boosted by high doses of each of the five mushroom extracts (207-289% increase for WBC and 153-175% for lymphocytes) with a significant increase in lysozyme activity (110-136% increase), NO concentration (159-232% increase) and cytokines as compared to the negative control group. Histopathological examination of the rats' spleen and thymus tissues has shown marked lymphocytic proliferation that was more obvious at the higher doses. In conclusion, our results showed that the five edible mushroom extracts revealed significant immunostimulatory effects preclinically particularly, at the higher doses (400 mg/kg) which can be considered the effective dose.

Alginate/κ-carrageenan oral microcapsules loaded with Agaricus bisporus polysaccharides MH751906 for natural killer cells mediated colon cancer immunotherapy.

The current study explores the effect of the extracted novel Mushroom polysaccharides and its formulation onto Alginate (Alg.)/kappa carrageenan microcapsules to exert immunotherapeutic effect upon activating gut resident natural killer cells (NK) against colon cancer. The extracted polysaccharides of Agaricus bisporus MH751906 was microcapsulated in Alg/κ-carrageenan microcapsules as an oral delivery system for colon cancer. The microcapsule is characterized by SEM, FTIR, Raman and TGA; and showed a superior acidic stability, controlled release, and thermal stability at high temperature with higher hydrogel swelling rate in colon-mimicking pH. Upon activation of human NK cells with microcapsules (ANK cells), a significant increase in CD16+CD56+ NK cell populations were recorded. These activated NK cells showed 74.09% cytotoxic effects against human colon cancer Caco-2 cells where majority of cancer cell populations arrested at G0/G1 phase leading to apoptosis. The apoptotic molecular mechanism induced by ANK cells on Caco-2 treated cells is through down regulations of both BCL2 and TGF surviving genes and up regulation in IkappaB-α gene expression. Therefore, this novel polysaccharides-alginate/κ-carrageenan microcapsules can be used as an oral targeted delivery system for colon cancer immunotherapy.

Antiviral, Cytotoxic, and Antioxidant Activities of Three Edible Agaricomycetes Mushrooms: Pleurotus columbinus, Pleurotus sajor-caju, and Agaricus bisporus.

In this study, we investigated aqueous extracts of three edible mushrooms: Agaricus bisporus (white button mushroom), Pleurotus columbinus (oyster mushroom), and Pleurotus sajor-caju (grey oyster mushroom). The extracts were biochemically characterized for total carbohydrate, phenolic, flavonoid, vitamin, and protein contents besides amino acid analysis. Triple TOF proteome analysis showed 30.1% similarity between proteomes of the two Pleurotus spp. All three extracts showed promising antiviral activities. While Pleurotus columbinus extract showed potent activity against adenovirus (Ad7, selectivity index (SI) = 4.2), Agaricus bisporus showed strong activity against herpes simplex II (HSV-2; SI = 3.7). The extracts showed low cytotoxicity against normal human peripheral blood mononuclear cells (PBMCs) and moderate cytotoxicity against prostate (PC3, DU-145); colorectal (Colo-205); cecum carcinoma (LS-513); liver carcinoma (HepG2); cervical cancer (HeLa); breast adenocarcinoma (MDA-MB-231 and MCF-7) as well as leukemia (CCRF-CEM); acute monocytic leukemia (THP1); acute promyelocytic leukemia (NB4); and lymphoma (U937) cell lines. Antioxidant activity was evaluated using 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) radical scavenging, 2,2'-Azinobis-(3-Ethylbenzthiazolin-6-Sulfonic Acid) ABTS radical cation scavenging, and oxygen radical absorbance capacity (ORAC) assays. The three extracts showed potential antioxidant activities with the maximum activity recorded for Pleurotus columbinus (IC50 µg/mL) = 35.13 ± 3.27 for DPPH, 13.97 ± 4.91 for ABTS, and 29.42 ± 3.21 for ORAC assays.

Proteome Analysis and In Vitro Antiviral, Anticancer and Antioxidant Capacities of the Aqueous Extracts of Lentinula edodes and Pleurotus ostreatus Edible Mushrooms.

In this study, we examined aqueous extracts of the edible mushrooms Pleurotus ostreatus (oyster mushroom) and Lentinula edodes (shiitake mushroom). Proteome analysis was conducted using LC-Triple TOF-MS and showed the expression of 753 proteins by Pleurotus ostreatus, and 432 proteins by Lentinula edodes. Bioactive peptides: Rab GDP dissociation inhibitor, superoxide dismutase, thioredoxin reductase, serine proteinase and lectin, were identified in both mushrooms. The extracts also included promising bioactive compounds including phenolics, flavonoids, vitamins and amino acids. The extracts showed promising antiviral activities, with a selectivity index (SI) of 4.5 for Pleurotus ostreatus against adenovirus (Ad7), and a slight activity for Lentinula edodes against herpes simplex-II (HSV-2). The extracts were not cytotoxic to normal human peripheral blood mononuclear cells (PBMCs). On the contrary, they showed moderate cytotoxicity against various cancer cell lines. Additionally, antioxidant activity was assessed using DPPH radical scavenging, ABTS radical cation scavenging and ORAC assays. The two extracts showed potential antioxidant activities, with the maximum activity seen for Pleurotus ostreatus (IC50 µg/mL) = 39.46 ± 1.27 for DPPH; 11.22 ± 1.81 for ABTS; and 21.40 ± 2.20 for ORAC assays. This study encourages the use of these mushrooms in medicine in the light of their low cytotoxicity on normal PBMCs vis à vis their antiviral, antitumor and antioxidant capabilities.

Rhizopus oryzae-Mediated Green Synthesis of Magnesium Oxide Nanoparticles (MgO-NPs): A Promising Tool for Antimicrobial, Mosquitocidal Action, and Tanning Effluent Treatment.

The metabolites of the fungal strain Rhizopus oryaze were used as a biocatalyst for the green-synthesis of magnesium oxide nanoparticles (MgO-NPs). The production methodology was optimized to attain the maximum productivity as follows: 4 mM of precursor, at pH 8, incubation temperature of 35 °C, and reaction time of 36 h between metabolites and precursor. The as-formed MgO-NPs were characterized by UV-Vis spectroscopy, TEM, SEM-EDX, XRD, DLS, FT-IR, and XPS analyses. These analytical techniques proved to gain crystalline, homogenous, and well-dispersed spherical MgO-NPs with an average size of 20.38 ± 9.9 nm. The potentiality of MgO-NPs was dose- and time-dependent. The biogenic MgO-NPs was found to be a promising antimicrobial agent against the pathogens including Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans with inhibition zones of 10.6 ± 0.4, 11.5 ± 0.5, 13.7 ± 0.5, 14.3 ± 0.7, and 14.7 ± 0.6 mm, respectively, at 200 µg mL-1. Moreover, MgO-NPs manifested larvicidal and adult repellence activity against Culex pipiens at very low concentrations. The highest decolorization percentages of tanning effluents were 95.6 ± 1.6% at 100 µg/ 100 mL after 180 min. At this condition, the physicochemical parameters of tannery effluents, including TSS, TDS, BOD, COD, and conductivity were reduced with percentages of 97.9%, 98.2%, 87.8%, 95.9%, and 97.3%, respectively. Moreover, the chromium ion was adsorbed with percentages of 98.2% at optimum experimental conditions.

Distribution of Extended-Spectrum ß-Lactamase (ESBL)-Encoding Genes among Multidrug-Resistant Gram-Negative Pathogens Collected from Three Different Countries.

The incidence of Extended-spectrum ß-lactamase (ESBL)-encoding genes (blaCTX-M and blaTEM) among Gram-negative multidrug-resistant pathogens collected from three different countries was investigated. Two hundred and ninety-two clinical isolates were collected from Egypt (n = 90), Saudi Arabia (n = 162), and Sudan (n = 40). Based on the antimicrobial sensitivity against 20 antimicrobial agents from 11 antibiotic classes, the most resistant strains were selected and identified using the Vitek2 system and 16S rRNA gene sequence analysis. A total of 85.6% of the isolates were found to be resistant to more than three antibiotic classes. The ratios of the multidrug-resistant strains for Egypt, Saudi Arabia, and Sudan were 74.4%, 90.1%, and 97.5%, respectively. Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa showed inconstant resistance levels to the different classes of antibiotics. Escherichia coli and Klebsiella pneumoniae had the highest levels of resistance against macrolides followed by penicillins and cephalosporin, while Pseudomonas aeruginosa was most resistant to penicillins followed by classes that varied among different countries. The isolates were positive for the presence of the blaCTX-M and blaTEM genes. The blaCTX-M gene was the predominant gene in all isolates (100%), while blaTEM was detected in 66.7% of the selected isolates. This work highlights the detection of multidrug-resistant bacteria and resistant genes among different countries. We suggest that the medical authorities urgently implement antimicrobial surveillance plans and infection control policies for early detection and effective prevention of the rapid spread of these pathogens.

Green Synthesis of Zinc Oxide Nanoparticles (ZnO-NPs) Using Arthrospira platensis (Class: Cyanophyceae) and Evaluation of their Biomedical Activities.

In this study, zinc oxide nanoparticles (ZnO-NPs) were successfully fabricated through the harnessing of metabolites present in the cell filtrate of a newly isolated and identified microalga Arthrospira platensis (Class: Cyanophyceae). The formed ZnO-NPs were characterized by UV-Vis spectroscopy, Fourier transform infrared (FT-IR), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS). Data showed the efficacy of cyanobacterial metabolites in fabricating spherical, crystallographic ZnO-NPs with a size ≈30.0 to 55.0 nm at a wavelength of 370 nm. Moreover, FT-IR analysis showed varied absorption peaks related to nanoparticle formation. XPS analysis confirms the presence of Zn(II)O at different varied bending energies. Data analyses exhibit that the activities of biosynthesized ZnO-NPs were dose-dependent. Their application as an antimicrobial agent was examined and formed clear zones, 24.1 ± 0.3, 21.1 ± 0.06, 19.1 ± 0.3, 19.9 ± 0.1, and 21.6 ± 0.6 mm, at 200 ppm against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Candida albicans, respectively, and these activities were reduced as the NPs concentration decreased. The minimum inhibitory concentration (MIC) values were determined as 50 ppm for S. aureus, 25 ppm for P. aeruginosa, and 12.5 ppm for B. subtilis, E. coli, and C. albicans. More interestingly, ZnO-NPs exhibit high in vitro cytotoxic efficacy against cancerous (Caco-2) (IC50 = 9.95 ppm) as compared with normal (WI38) cell line (IC50 = 53.34 ppm).

HBVsvp-Pulsed Dendritic Cell Immunotherapy Induces Th1 Polarization and Hepatitis B Virus-Specific Cytotoxic T Lymphocytes Production.

BACKGROUND: In chronic hepatitis B virus (CHB) patients, both dendritic cells (DCs) and T cells are functionally impaired and consequently the HBV-specific cellular immune responses are downregulated. The present study aims to investigate whether monocyte-derived DC (MoDCs)-pulsed-HBV subviral particles (HBVsvp) can polarize Th1 cells to induce HBV-specific cytotoxic T-lymphocytes (CTL) responses in CHB patients. METHODS AND MATERIALS: To this end, the human hepatoma HepG2.2.15 cell line was used to produce HBVsvp as a culturing system, and HBVsvp were concentrated for highly virus titer using the polyethylene glycol protocol. Peripheral blood mononuclear cells (PBMCs), collected from CHB patients and healthy donors, were differentiated into MoDCs and T cells. PBMCs-derived MoDCs were first pulsed with HBVsvp and then cultured with PBMCs-derived T cells. MoDCs and/or T subsets cells were identified for phenotypic activation by FACS analysis. The cytokine secretion of IL-4, IL-12, and IFN-γ in the culture supernatants was detected. RESULTS: The MoDCs were restored for their activation upon pulsing with HBVsvp in vitro, as identified by significantly overexpression of both CD86 and HLA-DR, and overproduction of IL-4 and IL-12. Furthermore, MoDCs-pulsed-HBVsvp induced Th1 frequencies and activated HBV-specific CTL to produce significantly highest amount of IFN-γ. Enhanced HBV-specific CTL led to strong cytolytic capacity against HepG2.2.15. CONCLUSION: Overall, our data suggest that in vitro activation of MoDCs with HBVsvp overcomes the functionally impaired DCs and T cells in CHB patients offering a promising tool for therapeutic or vaccine-based approaches against HBV.

Cloning, expression and nanodiscs assemble of recombinant HIV-1 gp41.

Structural studies of membrane proteins have been hurdled by their difficulty for expression in heterogeneous expression systems due to their intrinsically strong hydrophobicity and requirements for association with other cellular membranes. This study aims to design a construct for expression of membrane proteins. Because of its outstanding interest in HIV-1 vaccine design, transmembrane gp41 amino acid residue 662-723 was chosen as a representative membrane protein. Therefore, we constructed expression vectors for expression of gp41(662-723) alone (pET28a-gp41(662-723)) or coupled with a fusion partner: GB1 (pET30a-GB1-gp41(662-723)) and Trx (pET32a-Trx-gp41(662-723)). For enhancing protein expression, the expression plasmids were transformed into E. coli BL-21 (DE3), E. coli T7 Express lysY/Iq and E. coli Lemo21 (DE3). Interestingly, HIV-1 gp41(662-723) was expressed as a C-terminus fusion to the fusion partner Trx (Trx-gp41(662-723)) with an apparent molecular mass of 21.8 kDa. Trx-gp41(662-723) was overexpressed into E. coli T7 Express lysY/Iq by early induction as OD600 ~0.5 followed by incubation at 20 °C/overnight. Our data demonstrated that almost all recombinant Trx-gp41(662-723) was incorporated into lipid nanodiscs by slowing down the nanodiscs assembly process. Negative-stained electron micrographs revealed homogenous 10 nm Trx-gp41(662-723)-nanodiscs. While the neutralizing epitopes in the purified Trx-gp41(662-723) were accessible and recognizable by anti-MPER bNAbs, these epitopes became less accessibly exposed, particularly in the C-terminal region of MPER, after incorporation of Trx-gp41(662-723) into nanodiscs.

Antitumor effect of exopolysaccharide produced by Bacillus mycoides.

Recently, microbial exopolysaccharides (EPSs) have offered very large field for medical applications owing to their bioactive characteristics. This study aimed to obtain antitumor EPS and to optimize its production using different optimization approaches. Eighty EPSs-producing bacteria were obtained from mud samples. Isolate BS4 was selected as the most potent antitumor EPS-producer and identified as Bacillus mycoides BS4 using 16S rRNA gene sequence analysis. Cell viability and antitumor activity of produced EPS were investigated using microscopic examination and MTT assay. Interestingly, the produced EPS exhibited low cytotoxicity against normal cell baby hamster kidney (BHK) with IC50 at 254 µgml-1 while it exhibited an inhibitory effect against cancer cells of human hepatocellular carcinoma (HepG2) and Colorectal adenocarcinoma cells (Caco-2) with IC50 of 138 µgml-1 and 159 µgml-1, respectively. The purified EPS was characterized using Fourier transform infrared, gel permeation chromatography and gas chromatography-mass spectroscopy. It showed molecular weight of 1.90 × 104 Da and consists of galactose, mannose, glucose and glucuronic acid. The factors affecting EPS production were optimized using one-factor-at-a time and statistical optimization methods. The Placket-Burman (PB) design results indicated that sugarcane molasses, peptone and shaking conditions were the most significant variables, which were further optimized by Response Surface Methodology (RSM). The optimum conditions for EPS production were 8.0% (w/v) sugarcane molasses, 6 gL‒1 peptone and 300 rpm that produce 8.02gL‒1 of EPS. This indicates the potentiality of Bacillus mycoides BS4 for production of EPS with biomedical applications.

Scaling up of levan yield in Bacillus subtilis M and cytotoxicity study on levan and its derivatives.

This study focused on kinetics of levan yield by Bacillus subtilis M, in a 150 L stirred tank bioreactor under controlled pH conditions. The optimized production medium was composed of (g/L): commercial sucrose 100.0, yeast extract 2.0, K2HPO4 3.0 and MgSO4⋅7H2O 0.2; an increase in both carbohydrates consumption and cell growth depended on increasing the size of the stirred tank bioreactor from 16 L to 150 L. The highest levansucrase production (63.4 U/mL) and levan yield of 47 g/L was obtained after 24 h. Also, the specific levan yield (Yp/x) which reflects the cell productivity increased with the size increase of the stirred tank bioreactor and reached its maximum value of about 29.4 g/g cells. These results suggested that B. subtilis M could play an important role in levan yield on a large scale in the future. Chemical modifications of B. subtilis M crude levan (CL) into sulfated (SL), phosphorylated (PL), and carboxymethylated levans (CML) were done. The difference in CL structure and its derivatives was detected by FT-IR transmission spectrum. The cytotoxicity of CL and its derivatives were evaluated by HepGII, Mcf-7 and CaCo-2. In general most tested levans forms had no significant cytotoxicity effect. In fact, the carboxymethylated and phosphrylated forms had a lower anti-cancer effect than CL. On the other hand, SL had the highest cytotoxicity showing SL had a significant anti-cancer effect. The results of cytotoxicity and cell viability were statistically analyzed using three-way ANOVA.

Optimization of the Linker Length of Mannose-Cholesterol Conjugates for Enhanced mRNA Delivery to Dendritic Cells by Liposomes.

Liposomes (LPs) as commonly used mRNA delivery systems remain to be rationally designed and optimized to ameliorate the antigen expression of mRNA vaccine in dendritic cells (DCs). In this study, we synthesized mannose-cholesterol conjugates (MPn-CHs) by click reaction using different PEG units (PEG100, PEG1000, and PEG2000) as linker molecules. MPn-CHs were fully characterized and subsequently used to prepare DC-targeting liposomes (MPn-LPs) by a thin-film dispersion method. MPn-LPs loaded with mRNA (MPn-LPX) were finally prepared by a simple self-assembly method. MPn-LPX displayed bigger diameter (about 135 nm) and lower zeta potential (about 40 mV) compared to MPn-LPs. The in vitro transfection experiment on DC2.4 cells demonstrated that the PEG length of mannose derivatives had significant effect on the expression of GFP-encoding mRNA. MP1000-LPX containing MP1000-CH can achieve the highest transfection efficiency (52.09 ± 4.85%), which was significantly superior to the commercial transfection reagent Lipo 3K (11.47 ± 2.31%). The optimal DC-targeting MP1000-LPX showed an average size of 132.93 ± 4.93 nm and zeta potential of 37.93 ± 2.95 mV with nearly spherical shape. Moreover, MP1000-LPX can protect mRNA against degradation in serum with high efficacy. The uptake study indicated that MP1000-LPX enhanced mRNA expression mainly through the over-expressing mannose receptor (CD206) on the surface of DCs. In conclusion, mannose modified LPs might be a potential DC-targeting delivery system for mRNA vaccine after rational design and deserve further study on the in vivo delivery profile and anti-tumor efficacy.

Exopolysaccharide production from Bacillus velezensis KY471306 using statistical experimental design

Abstract Exopolysaccharide (EPS) biopolymers produced by microorganisms play a crucial role in the environment such as health and bio-nanotechnology sectors, gelling agents in food and cosmetic industries in addition to bio-flocculants in the environmental sector as they are degradable, nontoxic. This study focuses on the improvement of EPS production through manipulation of different culture and environmental conditions using response surface methodology (RSM). Plackett-Burman design indicated that; molasses, yeast extract and incubation temperature are the most effective parameters. Box-Behnken RSM indicated that; the optimum concentration for each parameter was 12% (w/v) for molasses, 6 g/L yeast extract and 30 °C for incubation temperature. The most potent bacterial isolate was identified as Bacillus velezensis KY498625. After production, EPS was extracted, purified using DEAE-cellulose, identified using Fourier transform infrared (FTIR), gel permeation chromatography (GPC) and gas chromatography-mass spectroscopy (GC-MS). The result indicated that; it has molecular weight 1.14 × 105 D consisting of glucose, mannose and galactose.

Exopolysaccharide production from Bacillus velezensis KY471306 using statistical experimental design.

Exopolysaccharide (EPS) biopolymers produced by microorganisms play a crucial role in the environment such as health and bio-nanotechnology sectors, gelling agents in food and cosmetic industries in addition to bio-flocculants in the environmental sector as they are degradable, nontoxic. This study focuses on the improvement of EPS production through manipulation of different culture and environmental conditions using response surface methodology (RSM). Plackett-Burman design indicated that; molasses, yeast extract and incubation temperature are the most effective parameters. Box-Behnken RSM indicated that; the optimum concentration for each parameter was 12% (w/v) for molasses, 6g/L yeast extract and 30°C for incubation temperature. The most potent bacterial isolate was identified as Bacillus velezensis KY498625. After production, EPS was extracted, purified using DEAE-cellulose, identified using Fourier transform infrared (FTIR), gel permeation chromatography (GPC) and gas chromatography-mass spectroscopy (GC-MS). The result indicated that; it has molecular weight 1.14×105D consisting of glucose, mannose and galactose.

Immune tolerance against HBV can be overcome in HBV transgenic mice by immunization with dendritic cells pulsed by HBVsvp.

In chronic Hepatitis B Virus (HBV) infection the function of dendritic cells (DC), T- and B-cells is impaired. DC vaccination is an option to overcome this. DC pulsed in vitro with HBV sub viral particles (HBVsvp) and used to immunize mice can activate HBV directed humoral and cellular immune responses. In the present study we vaccinated HBV transgenic mice as a model for chronic HBV infection and observed humoral and cellular immune responses. In these mice, the lacking immune response against HBV is mainly due to peripheral tolerance. HBVsvp, together with LPS as a co-activating molecule, were used for pulsing and in vitro activation of DC. HBV transgenic mice were injected with pulsed DC two times. Four weeks after DC vaccination humoral and cellular immune responses, viral antigen levels and liver histology were analyzed. DC vaccinated HBV-transgenic mice developed a strong HBV specific antibody and T-cell response after DC vaccination. Neither circulating HBV antigen levels nor viremia, however, were controlled. No liver damage was observed. These results demonstrate that in vitro activation of DC and loading with HBVsvp can overcome tolerance against HBV and reactivate B- and T-cell responses in HBV transgenic mice, but were not sufficient to lead to virus control in these mice. Vaccination using DC, the key players of cellular and humoral immunity, after in vitro reactivation promises to break tolerance against HBV and may help patients with chronic hepatitis B to clear the infection.

Dendritic cells can effectively be pulsed by HBVsvp and induce specific immune reactions in mice.

Eradication of chronic Hepatitis B virus (HBV) infection, marked by HBs seroconversion, is very rarely achieved by treatment with nucleoside and nucleotide analogs. Therapeutic cell based approaches, like interferon therapy, have a higher chance of seroconversion. Dendritic cells (DC) are key players in the cellular immune response and have been shown to play an important role in controlling HBV infection. In this study, the potential of ex vivo activated DC to induce specific immune responses against HBV was examined. DC derived from bone-marrow of BALB/c or C56BL/6 mice were pulsed with HBV subviral particles (HBVsvp), derived from the HepG2.2.15 cell line. HepG2.2.15 produces subviral particles consisting of the HBc and HBs proteins. Thus, the entire "viral surface" is presented to DC to induce an immune reaction. In vitro pulsation with HBVsvp successfully activated bone-marrow derived DC, demonstrated by FACS analysis showing increased MHCII, CD 86 and CCR-7. Immunization of mice, via subcutaneous injection of the activated DC, induced HBV specific immune reactions which were measured by ELISA, ELISPOT and T-cell proliferation analysis. Vaccination with ex vivo activated DC may be a promising tool for therapeutic or prophylactic approaches against the Hepatitis B virus.