Clostridioides difficile Flagellin Activates the Intracellular NLRC4 Inflammasome.

Clostridioides difficile (C. difficile), is a major cause of nosocomial diarrhea and colitis. C. difficile flagellin FliC contributes toxins to gut inflammation by interacting with the immune Toll-like receptor 5 (TLR5) to activate nuclear factor-kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways. Flagella of intracellular pathogens can activate the NLR family CARD domain-containing protein 4 (NLRC4) inflammasome pathway. In this study, we assessed whether flagellin of the extracellular bacterium C. difficile internalizes into epithelial cells and activates the NLRC4 inflammasome. Confocal microscopy showed internalization of recombinant green fluorescent protein (GFP)-FliC into intestinal Caco-2/TC7 cell line. Full-length GFP-FliC activates NLRC4 in Caco-2/TC7 cells in contrast to truncated GFP-FliC lacking the C-terminal region recognized by the inflammasome. FliC induced cleavage of pro-caspase-1 into two subunits, p20 and p10 as well as gasdermin D (GSDMD), suggesting the caspase-1 and NLRC4 inflammasome activation. In addition, colocalization of GFP-FliC and pro-caspase-1 was observed, indicating the FliC-dependent NLRC4 inflammasome activation. Overexpression of the inflammasome-related interleukin (interleukin (IL)-1ß, IL-18, and IL-33) encoding genes as well as increasing of the IL-18 synthesis was detected after cell stimulation. Inhibition of I-kappa-B kinase alpha (IKK-α) decreased the FliC-dependent inflammasome interleukin gene expression suggesting a role of the NF-κB pathway in regulating inflammasome. Altogether, these results suggest that FliC internalizes into the Caco-2/TC7 cells and activates the intracellular NLRC4 inflammasome thus contributing to the inflammatory process of C. difficile infection.

A dye-affinity cryogel membrane for malate dehydrogenase purification from Saccharomyces cerevisiae.

Cibacron blue F3GA functionalized poly(hydroxyethyl methacrylate) cryogel membranes were prepared and applied for a simple purification of malate dehydrogenase (MDH) from crude extract of Saccharomyces cerevisiae. Swelling tests, scanning electron microscopy, surface area measurements and Fourier transform infrared (FTIR) spectroscopy techniques were used for the characterization of dye-affinity cryogel membranes. Following cell homogenization and extraction, MDH was purified using the dye-affinity cryogel membranes at a high yield of 80.5% with 54-fold purification. Maximum MDH adsorption amount was determined to be 267.7 mg/g of membranes at pH 7.4, 25 °C and a flow rate of 1.0 mL/min. Interestingly, the cryogel membranes were used for several purification runs without any significant decrease in MDH adsorption capacity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was carried out to assess the purity of the eluted MDH. The obtained results highlight the dye-affinity cryogel membranes as powerful dye affinity adsorbents for MDH purification from S. cerevisiae.

Enzyme-Initiated Free-Radical Polymerization of Molecularly Imprinted Polymer Nanogels on a Solid Phase with an Immobilized Radical Source.

An enzyme-mediated synthetic approach is described for the preparation of molecularly imprinted polymer nanoparticles (MIP-NPs) in aqueous media. Horseradish peroxidase (HRP) was used to initiate the polymerization of methacrylate or vinyl monomers and cross-linkers by catalyzing the generation of free radicals. To prevent entrapment of the enzyme in the cross-linked polymer, and to enable it to be reused, HRP was immobilized on a solid support. MIPs based on 4-vinylpyridine and 1,4-bis(acryloyl)piperazine for the recognition of 2,4-dichlorophenoxyacetic acid (2,4-D) and salicylic acid were synthesized in an aqueous medium. MIPs for the protein trypsin were also synthesized. MIP nanoparticles with sizes between 50 and 300 nm were obtained with good binding properties, a good imprinting effect, and high selectivity for the target molecule. The reusability of immobilized HRP for MIP synthesis was shown for several batches.

Synthesis of hydroxyethyl-methacrylate-(L)-histidine methyl ester cryogels. Application on the separation of bovine immunoglobulin G.

In this study cryogels based 2-hydroxyethyl methacrylate (HEMA) functionalized with N-methacryloyl-L-histidine methyl ester (MAH) were synthesized and used for the adsorption and separation of bovine IgG. Two series of cryogels functionalized with 5 and 10 mg of MAH as pseudobioaffinity ligand were prepared and characterized by swelling test, FTIR and SEM analysis. The adsorption efficiency of the bovine immunoglobulin into cryogels is discussed with respect to the following chromatographic parameters: pH, flow rate, initial IgG concentration, adsorption time and ionic strength. Our results show good adsorption of bovine immunoglobulin under mild separation conditions at pH 7.4. The maximum binding capacity was determined (32.4 mg/g of cryogel) and demonstrates the efficiency of the used cryogels. This efficacy is clearly seen upon increasing the maximum binding capacity from 23.2 mg (obtained with cryogels with 5 mg MAH) to 32.4 mg/g (for cryogel with 10 mg MAH ligand concentration). The purity of separated fractions was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Together our observations highlights poly (HEMA-MAH) as an efficient adsorbent for bovine immunoglobulins G separation.

[PHEMA/PEI]-Cu(II) based immobilized metal affinity chromatography cryogels: Application on the separation of IgG from human plasma.

The immobilized metal-affinity chromatography (IMAC) has gained significant interest as a widespread separation and purification tool for therapeutic proteins, nucleic acids and other biological molecules. The enormous potential of IMAC for proteins with natural surface exposed-histidine residues and for recombinant proteins with histidine clusters. Cryogels as monolithic materials have recently been proposed as promising chromatographic adsorbents for the separation of biomolecules in downstream processing. In the present study, IMAC cryogels have been synthesized and utilized for the adsorption and separation of immunoglobulin G (IgG) from IgG solution and whole human plasma. For this purpose, Cu(II)-ions were coupled to poly(hydroxyethyl methacrylate) PHEMA using poly(ethylene imine) (PEI) as the chelating ligand. In this study the cryogels formation optimized by the varied proportion of PEI from 1% to 15% along with different amounts of Cu (II) as chelating metal. The prepared cryogels were characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, and thermogravimetric analysis. The [PHEMA/PEI]-Cu(II) cryogels were assayed for their capability to bind the human IgG from aqueous solutions. The IMAC cryogels were found to have high affinity toward human IgG. The adsorption of human IgG was investigated onto the PHEMA/PEI cryogels with (10% PEI) and the concentration of Cu (II) varied as 10, 50, 100 and 150 mg/L. The separation of human IgG was achieved in one purification step at pH7.4. The maximum adsorption capacity was observed at the [PHEMA/PEI]-Cu(II) (10% PEI) with 72.28 mg/g of human IgG. The purification efficiency and human IgG purity were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Effects of methanol extracts from roots, leaves, and fruits of the Lebanese strawberry tree (Arbutus andrachne) on cardiac function together with their antioxidant activity.

CONTEXT: Several plant-derived natural products have been used in clinical phase for applications in neurological, cardiovascular, and inflammatory diseases. Arbutus andrachne L. (Ericacea) is an evergreen shrub native to the Mediterranean region. Traditionally, the fruits and leaves of Arbutus tree are well known and used as antiseptics, diuretics, blood tonic, and laxatives. OBJECTIVE: Data regarding the biological effects of compounds derived from the Lebanese Arbutus andrachne are not available. In the present work, we studied the antioxidant activity of methanol extracts of leaves, fruits, and roots of the plant against electrolysis; together with their effects on the cardiodynamics of isolated perfused rabbit hearts. MATERIALS AND METHODS: In vitro electrolysis of the different root, leaves, and fruits methanol extracts was evaluated by the amount of free radicals that has been reduced by increasing the concentration of root extracts ranging from 0.5 to 2 mg after 1, 2, 3, and 4 min. Left ventricular pressure (LVP), heart rate (HR), and coronary flow (CR) were investigated in isolated rabbit heart after administration of 0.5, 1, 2, and 2 mg of each methanol extracts plotted against time (0, 0.5, 1.5, 5, and 10 min), according to the Langendorff method. Lipid peroxidation study was performed by the colorimetric method on myocard tissue after incubation with 500 µl of the different methanol extracts. The amount of MDA was determined at 500 nm absorbance after 5 min incubation. RESULTS: Among the different methanol extracts, the roots showed the highest in vitro antioxidant activity, particularly observed at concentration of 2 mg which completely inhibits free radical generation after 4 min. LVP decreases by 32% at the dose of 2 mg of root extracts after 5 min. No significant effect was observed by the three tested extracts on the heart rate. The three methanol extracts did not show any significant effect on the coronary flow. Moreover, the roots show an increase in the coronary flow at a concentration of 1 and 2 mg/ml during 1 min. Electrolysis on heart tissue treated with the roots extracts shows a decrease in the MDA level from 70.51 ± 6.71 to 48.58 ± 4.15 nmole/g of tissue. DISCUSSION AND CONCLUSION: Methanol extracts of the roots possess antihypertensive effect that may result from its ability to decrease the LVP together with its protective role by inhibiting free radical generation and significantly decreasing the MDA level of heart tissue.

Affinity composite cryogel discs functionalized with Reactive Red 120 and Green HE 4BD dye ligands: application on the separation of human immunoglobulin G subclasses.

Naturally produced by the human immune system, immunoglobulin nowadays is widely used for in vivo and in vitro purposes. The increased needs for pure immunoglobulin have prompted researchers to find new immunoglobulin chromatographic separation processes. Cryogels as chromatographic adsorbents, congregate several mechanical features including good compatibility, large pore structure, flexibility, short diffusion pathway and stability. These different characteristics make them a good alternative to conventional chromatographic methods and allowing their potential use in separation technology. In the present study, two sets of poly(2-hydroxyethyl methacrylate) (PHEMA) based beads were prepared and functionalized with Reactive Red 120 (RR) and Reactive Green HE 4BD (RG) dyes, and then embedded into supermacroporous cryogels. The morphology, physical and chemical features of the prepared bead embedded composite cryogel discs (CCDs) were performed by scanning electron microscopy (SEM), swelling test, elemental analysis and Fourier transform infrared spectroscopy (FTIR). The results showed that the embedded composite cryogel discs have a specific surface area of 192.0 m(2)/g with maximum adsorption capacity of HIgG 239.8 mg/g for the RR functionalized CCD and 170 mg/g for RG functionalized CCD columns, both at pH 6.2.

Histidine based adsorbents for selective removal of monoclonal immunoglobulin IgM antibodies from Waldenstrom's macroglobulinemia patient sera: a preliminary study.

Waldenstrom's macroglobulinemia (WM) is considered by the Revised European American Lymphoma (REAL) and World Health Organization (WHO) as a clinical lympho plasmacytic syndrome associated with high monoclonal (IgM) secretion. The hyper viscosity syndrome is associated with several clinical disorders of monoclonal IgM. Patients with clinical symptoms of hyper viscosity should be treated with plasma pheresis, which is limited by its non-selective removal of all plasma components. These limitations have steered efforts to find a more specific removal according to clinical needs and avoiding plasma components replacement. Removal by specific adsorption is the most powerful selective apheresis technique. The active adsorbed ligand is covalently bound to an insoluble matrix through which plasma is passed. Amino acids have been introduced as ligands in clinical apheresis for the removal of auto antibodies associated with autoimmune diseases. The present preliminary study describes the binding of monoclonal IgM antibodies from sera of patients with WM, on histidine immobilized to activated sepharose. The advantages of efficient binding and elution, suggest histidine adsorbents as prospective clinical means suitable for the removal of monoclonal IgM from sera of patients diagnosed with WM. The advantages of efficient adsorption and elution, non toxicity of histidine, good selectivity, good stability, as well as their low cost strongly suggest histidine adsorbents as prospective clinical means suitable for the removal of monoclonal IgM from sera of patients diagnosed with WM.

Phenylalanine containing hydrophobic nanospheres for antibody purification.

In this study, novel hydrophobic nanospheres with an average size of 158 nm utilizing N-methacryloyl-(L)-phenylalanine methyl ester (MAPA) as a hydrophobic monomer were produced by surfactant free emulsion polymerization of 2-hydroxyethyl methacrylate (HEMA) and MAPA conducted in an aqueous dispersion medium. MAPA was synthesized using methacryloyl chloride and L-phenylalanine methyl ester. Specific surface area of the nonporous nanospheres was found to be 1874 m2/g. Poly(HEMA-MAPA) nanospheres were characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Average particle size, size distribution, and surface charge measurements were also performed. Elemental analysis of MAPA for nitrogen was estimated as 0.42 mmol/g polymer. Then, poly(HEMA-MAPA) nanospheres were used in the adsorption of immunoglobulin G (IgG) in batch system. Higher adsorption values (780 mg/g) were obtained when the poly (HEMA-MAPA) nanospheres were used from both aqueous solutions and human plasma. The adsorption phenomena appeared to follow a typical Langmuir isotherm. It was observed that IgG could be repeatedly adsorbed and desorbed without significant loss in adsorption amount. These findings show considerable promise for this material as a hydrophobic support in industrial processes.