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Analytical Chemistry Sep 2017Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE),...
Given the wide adoption of polydimethylsiloxane (PDMS) for the rapid fabrication of microfluidic networks and the utility of polyacrylamide gel electrophoresis (PAGE), we develop a technique for fabrication of PAGE molecular sieving gels in PDMS microchannel networks. In developing the fabrication protocol, we trade-off constraints on materials properties of these two polymer materials: PDMS is permeable to O and the presence of O inhibits the polymerization of polyacrylamide. We present a fabrication method compatible with performing PAGE protein separations in a composite PDMS-glass microdevice, that toggles from an "enclosed" microchannel for PAGE and blotting to an "open" PA gel lane for immunoprobing and readout. To overcome the inhibitory effects of O, we coat the PDMS channel with a 10% benzophenone solution, which quenches the inhibiting effect of O when exposed to UV, resulting in a PAGE-in-PDMS device. We then characterize the PAGE separation performance. Using a ladder of small-to-mid mass proteins (Trypsin Inhibitor (TI); Ovalbumin (OVA); Bovine Serum Albumin (BSA)), we observe resolution of the markers in <60 s, with separation resolution exceeding 1.0 and CVs of 8.4% for BSA-OVA and 2.4% for OVA-TI, with comparable reproducibility to glass microdevice PAGE. We show that benzophenone groups incorporated into the gel through methacrylamide can be UV-activated multiple times to photocapture protein. PDMS microchannel network is reversibly bonded to a glass slide allowing direct access to separated proteins and subsequent in situ diffusion-driven immunoprobing and total protein Sypro red staining. We see this PAGE-in-PDMS fabrication technique as expanding the application and use of microfluidic PAGE without the need for a glass microfabrication infrastructure.
Topics: Adoption; Animals; Cattle; Dimethylpolysiloxanes; Electrophoresis, Polyacrylamide Gel; Immunoblotting; Microfluidic Analytical Techniques; Ovalbumin; Particle Size; Serum Albumin, Bovine; Trypsin Inhibitors
PubMed: 28825964
DOI: 10.1021/acs.analchem.7b02406 -
International Journal For Parasitology Feb 2011Entamoeba histolytica, an intestinal amoeba that causes dysentery and liver abscesses, acquires nutrients by engulfing bacteria in the colonic lumen and phagocytoses...
Entamoeba histolytica, an intestinal amoeba that causes dysentery and liver abscesses, acquires nutrients by engulfing bacteria in the colonic lumen and phagocytoses apoptotic cells during tissue invasion. In preliminary studies to identify ligands that stimulate amoebic phagocytosis, we used ovalbumin immobilized on latex particles as a potential negative control protein. Surprisingly, ovalbumin strongly stimulated E. histolytica particle uptake. Experiments using highly purified ovalbumin confirmed the specificity of this finding. The mechanism of particle uptake was actin-dependent, and the Entamoeba phagosome marker amoebapore A localised to ovalbumin-bead containing vacuoles. The most well described amoebic receptor is a Gal/GalNAc-specific lectin, but d-galactose had no effect on ovalbumin-stimulated phagocytosis. Ovalbumin has a single N-glycosylation site (Asn(292)) and is modified with oligomannose and hybrid-type oligosaccharides. We used both trifluoromethanesulfonic acid and N-glycanase to deglycosylate ovalbumin and tested the effect. Both methods substantially reduced the stimulatory effect of ovalbumin. Biotinylated ovalbumin bound the surface of fixed E. histolytica trophozoites saturably; furthermore, denatured ovalbumin and native ovalbumin both specifically inhibited ovalbumin-biotin binding, but deglycosylated ovalbumin had no effect. Collectively, these data suggest that E. histolytica has a previously unrecognised surface lectin activity that binds to carbohydrates on ovalbumin and stimulates phagocytosis.
Topics: Carbohydrate Metabolism; Entamoeba histolytica; Humans; Ion Channels; Lectins; Microspheres; Ovalbumin; Phagocytosis; Phagosomes; Protein Binding; Protozoan Proteins
PubMed: 20807536
DOI: 10.1016/j.ijpara.2010.07.011 -
International Journal of Biological... Sep 2017The interactions of tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) with ovalbumin (OVA), the main allergen protein of egg white, were investigated...
The interactions of tetracycline (TC), oxytetracycline (OTC) and chlortetracycline (CTC) with ovalbumin (OVA), the main allergen protein of egg white, were investigated by molecular spectroscopy and electrophoresis at three pH conditions (1.5, 4.6 and 7.4). Molecular and synchronous fluorescence, UV-vis spectroscopy, electrophoresis and H NMR were used to study the interaction process. Tetracyclines interact with ovalbumin fluorescence by a static quenching mechanism with non-fluorescent complex formation changing the native protein structure. The binding constant (K) ranged from 2.11×10 to 58.4×10Lmol, and corresponding thermodynamic parameters were measured at different temperatures and pH values. The binding process was spontaneous (ΔG<0), and the magnitude of the interaction increased in the following order: TC
Topics: Allergens; Animals; Egg White; Electrophoresis; Ovalbumin; Protein Binding; Spectrum Analysis; Tetracyclines
PubMed: 28428126
DOI: 10.1016/j.ijbiomac.2017.04.052 -
Methods and Findings in Experimental... 2003It has been suggested that epithelium destruction, mucus secretion, edema and bronchoconstriction may play a role in asthma pathogenesis. Additionally, histamine,...
It has been suggested that epithelium destruction, mucus secretion, edema and bronchoconstriction may play a role in asthma pathogenesis. Additionally, histamine, serotonine, leukotrienes and other mediators are released and free oxygen radicals are produced during bronchoconstriction. We studied the role of antioxidants on the treatment of asthma by testing alpha-tocopherol (vitamin E; 5, 25, 50 and 100 mg/kg/day i.p.), a scavenger of oxygen radicals, on male Guinea pigs. The animals were sensitized by injecting ovalbumin. After sensitization, isolated tracheal preparations were studied in vitro. The effects of carbachol 10(-4) M, ovalbumin 10(-3) M and vitamin E (10(-5) M, 10(-4) M, 10(-3) M concentrations) were evaluated in an isolated organ bath. It was observed that alpha-tocopherol reduced the contractile effects of ovalbumin and carbachol. The 5, 25 and 50 mg/kg/day doses of vitamin E were injected intraperitoneally to Guinea pigs that were sensitized with ovalbumin. We observed significant differences between the contractile responses to carbachol and vitamin E with carbachol. These treatments significantly reduced the contractility of tracheal preparations (p < 0.001). There was no significant difference with the 100 mg/kg/day i.p. dose of vitamin E in the contractile response to carbachol E (p > 0.05). The upper part of the tracheal preparations was the most sensitive region to the contractile effect of 10(-3) M ovalbumin in all groups (p < 0.001). The contractile response to 10(-3) M ovalbumin was reduced by 25 and 50 mg/kg/day doses of vitamin E (p < 0.001).
Topics: Animals; Bronchial Spasm; Guinea Pigs; Immunization; In Vitro Techniques; Injections, Intraperitoneal; Male; Muscle Contraction; Ovalbumin; Trachea; Vitamin E
PubMed: 12690703
DOI: 10.1358/mf.2003.25.1.772544 -
The Journal of Cell Biology Sep 1971Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium; these cells...
Administration of estrogen (E) to immature chicks triggers the cytodifferentiation of tubular gland cells in the magnum portion of the oviduct epithelium; these cells synthesize the major egg-white protein, ovalbumin. Electron microscopy and immunoprecipitation of ovalbumin from oviduct explants labeled with radioactive amino acids in tissue culture were used to follow and measure the degree of tubular gland cell cytodifferentiation. Ovalbumin is undetectable in the unstimulated chick oviduct and in oviducts of chicks treated with progesterone (P) for up to 5 days. Ovalbumin synthesis is first detected 24 hr after E administration, and by 5 days it accounts for 35% of the soluble protein being synthesized. Tubular gland cells begin to synthesize ovalbumin before gland formation which commences after 36 hr of E treatment. When E + P are administered together there is initially a synergistic effect on ovalbumin synthesis, however, after 2 days ovalbumin synthesis slows and by 5 days there is only 1/20th as much ovalbumin per magnum as in the E-treated controls. Whereas the magnum wet weight doubles about every 21 hr with E alone, growth stops after 3 days of E + P treatment. Histological and ultrastructural observations show that the partially differentiated tubular gland cells resulting from E + P treatment never invade the stroma and form definitive glands, as they would with E alone. Instead, these cells appear to transform into other cell types-some with cilia and some with unusual flocculent granules. We present a model of tubular gland cell cytodifferentiation and suggest that a distinct protodifferentiated stage exists. P appears to interfere with the normal transition from the protodifferentiated state to the mature tubular gland cell.
Topics: Animals; Cell Differentiation; Chickens; Cilia; Culture Techniques; Cytoplasmic Granules; Electrophoresis, Disc; Endoplasmic Reticulum; Epithelium; Estrogens; Female; Histocytochemistry; Inclusion Bodies; Methods; Microscopy, Electron; Models, Biological; Morphogenesis; Organ Size; Ovalbumin; Oviducts; Precipitin Tests; Progesterone; Protein Biosynthesis; Time Factors
PubMed: 4329151
DOI: 10.1083/jcb.50.3.598 -
Journal of Pharmacy & Pharmaceutical... 2014The objective of this work was to evaluate the effect in the immune response produced by CpG oligodeoxynucleotides (ODN) co-encapsulated with the antigen ovalbumin (OVA)...
PURPOSE
The objective of this work was to evaluate the effect in the immune response produced by CpG oligodeoxynucleotides (ODN) co-encapsulated with the antigen ovalbumin (OVA) within poly(lactic-co-glycolic) acid (PLGA) 502 and 752 microparticles (MP).
METHODS
MP were prepared by blending 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) with PLGA and Total Recirculation One Machine System (TROMS) technology and contained OVA along with CpG sequences associated to DOTAP. After confirming the integrity of both encapsulated molecules, BALB/c mice were immunized with the resulting MP and OVA-specific antibodies and cytokine production were assessed in order to determine the immunological profile induced in mice.
RESULTS
One m near non-charged MP co-encapsulated very efficiently both OVA and CpG ODN. The release of both OVA and CpG was slow and incomplete irrespective of polymer. The results of the immune response induced in BALB/c mice indicated that, depending on the PLGA polymer used, co-encapsulation did not improve the immunogenicity of the antigen, compared either with the simply co-administration of both antigen and CpG, or with the microencapsulated antigen. Thus, mice immunized with OVA associated to PLGA 756 displayed an IgG2a characterized response which was biased to an IgG1 profile in case of CpG co-encapsulation. On the contrary, the co-encapsulation of CpG with OVA into PLGA 502 significantly improved the isotype shifting in comparison with the one showed by mice immunized with OVA loaded PLGA 502.
CONCLUSION
This study underlines the importance of MP characteristics to fully exploit simultaneous antigen and CpG ODN particulate delivery as effective vaccine construct.This article is open to POST-PUBLICATION REVIEW. Registered readers (see "For Readers") may comment by clicking on ABSTRACT on the issue's contents page.
Topics: Animals; Antibody Formation; Antigens; Female; Immunization; Lactic Acid; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Microspheres; Oligodeoxyribonucleotides; Ovalbumin; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Vaccines
PubMed: 25579433
DOI: 10.18433/j33892 -
British Journal of Cancer Apr 2023Dendritic cell (DC) vaccines for cancer therapy offer the possibility to let the patient's own immune system kill cancer cells. However, DC vaccines have shown less...
BACKGROUND
Dendritic cell (DC) vaccines for cancer therapy offer the possibility to let the patient's own immune system kill cancer cells. However, DC vaccines have shown less efficacy than expected due to failure to induce cancer cell killing and by activating T regulatory cells.
METHODS
We tested if inhibition of signalling via WASp and Arp2/3 using the small molecule CK666 would enhance DC-mediated killing of tumour cells in vitro and in vivo.
RESULTS
Using CK666 during the ex vivo phase of antigen processing of ovalbumin (OVA), murine and human DCs showed decreased phagosomal acidification, indicating activation of the cross-presentation pathway. When compared to untreated DCs, DCs treated with CK666 during uptake and processing of OVA-induced increased proliferation of OVA-specific CD8 OT-I T cells in vitro and in vivo. Using the aggressive B16-mOVA melanoma tumour model, we show that mice injected with CK666-treated DCs and OVA-specific CD8 OT-I T cells showed higher rejection of B16 melanoma cells when compared to mice receiving non-treated DCs. This resulted in the prolonged survival of tumour-bearing mice receiving CK666-treated DCs. Moreover, combining CK666-treated DCs with the checkpoint inhibitor anti-PD1 further prolonged survival.
CONCLUSION
Our data suggest that the small molecule inhibitor CK666 is a good candidate to enhance DC cross-presentation for cancer therapy.
Topics: Mice; Animals; Humans; Cross-Priming; CD8-Positive T-Lymphocytes; Dendritic Cells; Antigen Presentation; Ovalbumin; Vaccines; Mice, Inbred C57BL
PubMed: 36631633
DOI: 10.1038/s41416-022-02135-4 -
Poultry Science Mar 2019In addition to small amounts of minerals and carbohydrates, most of the dry matter of chicken egg white is protein, making egg white an ideal resource for obtaining food...
In addition to small amounts of minerals and carbohydrates, most of the dry matter of chicken egg white is protein, making egg white an ideal resource for obtaining food proteins. Ovalbumin (OVA), which accounts for more than 50% of the total egg white protein, is one of the most widely studied food proteins due to its multiple functional properties, and it has also been used as a model protein molecule in many research fields. The objective of this study was to develop a simple and rapid method for the purification of OVA from egg whites on large scale. First, OVA was separated from ovomucin, ovotransferrin, and ovomucoid by polyethylene glycol (PEG) concentration, using the following optimal parameters: the PEG concentration was 15%, the pH was 6.5, the salt concentration was 100 mmol/L, and the operating temperature was 10°C. The OVA-rich supernatant obtained from PEG precipitation was further purified by isoelectric precipitation at a pI of 4.5 and a temperature of 4°C, and the purified OVA was obtained with at a purity of 95.1% by HPLC, with a yield of 46.4%. After the extraction of OVA, the PEG solution was vacuum dried and then utilized cyclically in the PEG precipitation steps. The whole purification process could be finished within 2 to 3 h at a scale of several kilograms of egg white. This method has the advantages of rapidity, simplicity, low cost, and ease of scalability.
Topics: Animals; Chemical Precipitation; Chickens; Egg White; Ovalbumin; Polyethylene Glycols
PubMed: 30184130
DOI: 10.3382/ps/pey402 -
The Journal of Biological Chemistry Jul 2018Amyloids are associated with many neurodegenerative diseases, motivating investigations into their structure and function. Although not linked to a specific disease,...
Amyloids are associated with many neurodegenerative diseases, motivating investigations into their structure and function. Although not linked to a specific disease, albumins have been reported to form many structural aggregates. We were interested in investigating host immune responses to amyloid fibrils assembled from the model protein ovalbumin. Surprisingly, upon subjecting ovalbumin to standard denaturing conditions, we encountered giant protein nanosheets harboring amyloid-like features and hypothesized that these nanosheets might have potential in clinical or therapeutic applications. We found that the nanosheets, without the administration of any additional adjuvant, evoked a strong antibody response in mice that was higher than that observed for native ovalbumin. This suggests that amyloid nanosheets have a self-adjuvanting property. The nanosheet-induced immune response was helper T cell 2 (Th2) biased and negligibly inflammatory. While testing whether the nanosheets might form depots for the sustained release of precursor proteins, we did observe release of ovalbumin that mimicked the conformation of native protein. Moreover, the nanosheets could load the anticancer drug doxorubicin and release it in a slow and sustained manner. Taken together, our results suggest that amyloid nanosheets should be further investigated as either an antigen delivery vehicle or a multifunctional antigen and drug co-delivery system, with potential applications in simultaneous immunotherapy and chemotherapy.
Topics: Adjuvants, Immunologic; Amyloid; Animals; Antibiotics, Antineoplastic; Antibody Formation; Delayed-Action Preparations; Doxorubicin; Drug Liberation; Female; Immunization; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Nanostructures; Ovalbumin; Protein Denaturation; Th2 Cells
PubMed: 29853634
DOI: 10.1074/jbc.RA118.002550 -
Journal of Autoimmunity Nov 2010A major emphasis of our studies has been on developing a better understanding of how and why the skin serves as a target for immune reactions as well as how the skin... (Review)
Review
A major emphasis of our studies has been on developing a better understanding of how and why the skin serves as a target for immune reactions as well as how the skin evades becoming a target for destruction. For these studies we developed transgenic mice that express a membrane-tethered form of a model self antigen, chicken ovalbumin (mOVA), under the control of a keratin 14 (K14) promoter. K14-mOVA transgenic mice that express OVA mRNA and protein in the epithelia have been assessed for their immune responsiveness to OVA and are being used as targets for T cells obtained from OT-1 transgenic mice whose CD8+ T cells carry a Vα2/Vβ5-transgenic T cell receptor with specificity for the OVA(257-264)-peptides (OVAp) in association with class I MHC antigens. Some of the K14-mOVA transgenic mice develop a graft-versus-host-like disease (GvHD) when the OT-1 cells are injected while others appear to be tolerant to the OT-1 cells. We found that γc cytokines, especially IL-15, determine whether autoimmunity or tolerance ensues in K14-mOVA Tg mice. We also developed transgenic mice that express soluble OVA under the control of a K14 promoter (K14-sOVA) that die within 5-8 days after adoptive transfer of OT-1 cells and identified these mice as a model for more acute GvHD-like reactions. Spontaneous autoimmunity occurs when these K14-sOVA mice are crossed with the OT-I mice. In contrast, we found that preventive or therapeutic OVAp injections induced a dose-dependent increase in survival. In this review the characterization of 5 strains of K14-OVATg mice and underlying mechanisms involved in autoimmune reactions in these Tg mice are discussed. We also describe a strategy to break tolerance and describe how the autoimmunity can be obviated using OVAp. Finally, a historical overview of using transgenic mice to assess the mechanisms of tolerance is also provided.
Topics: Animals; Autoantigens; Autoimmunity; CD8-Positive T-Lymphocytes; Disease Models, Animal; Graft vs Host Disease; Humans; Immune Tolerance; Keratin-14; Mice; Mice, Transgenic; Ovalbumin; Promoter Regions, Genetic; Receptors, Antigen, T-Cell; Skin
PubMed: 20655706
DOI: 10.1016/j.jaut.2010.06.007