BACKGROUND: Chronic kidney disease is a severe threat to human health with no ideal treatment strategy. Mature mammalian kidneys have a fixed number of nephrons, and regeneration is difficult once they are damaged. For this reason, developing an efficient approach to achieve kidney regeneration is necessary. The technology of the combination of decellularized kidney scaffolds with stem cells has emerged as a new strategy; however, in previous studies, the differentiation of stem cells in decellularized scaffolds was insufficient for functional kidney regeneration, and many problems remain. METHODS: We used 0.5% sodium dodecyl sulfate (SDS) to produce rat kidney decellularized scaffolds, and induce adipose-derived stem cells (ADSCs) into intermediate mesoderm by adding Wnt agonist CHIR99021 and FGF9 in vitro. The characteristics of decellularized scaffolds and intermediate mesoderm induced from adipose–derived stem cells were identified. The scaffolds were recellularized with ADSCs and intermediate mesoderm cells through the renal artery and ureter. After cocultured for 10 days, cells adhesion and differentiation was evaluated. RESULTS: Intermediate mesoderm cells were successfully induced from ADSCs and identified by immunofluorescence and Western blotting assays (OSR1 + , PAX2 +). Immunofluorescence showed that intermediate mesoderm cells differentiated into tubular-like (E-CAD + , GATA3 +) and podocyte-like (WT1 +) cells with higher differentiation efficiency than ADSCs in the decellularized scaffolds. Comparatively, this phenomenon was not observed in induced intermediate mesoderm cells cultured in vitro. CONCLUSION: In this study, we demonstrated that intermediate mesoderm cells could be induced from ADSCs and that they could differentiate well after cocultured with decellularized scaffolds.
Animals ; Blotting, Western ; Fluorescent Antibody Technique ; Humans ; In Vitro Techniques ; Kidney ; Mesoderm ; Nephrons ; Rats ; Regeneration ; Renal Artery ; Renal Insufficiency, Chronic ; Sodium Dodecyl Sulfate ; Stem Cells ; Ureter

BACKGROUND: Black tiger shrimp Penaeus monodon is one of the common causes of shellfish allergy that is increasing worldwide. One of the important problems in the management of shellfish allergy is the lack of accurate diagnostic assay because the biological and immunological properties of allergens in black tiger shrimp have not been well characterized. This study aims to detect proteins with the ability to bind and cross-link immunoglobulin E (IgE) from black tiger shrimp by enzyme-linked immunosorbent assay (ELISA), Western blot, and a humanized rat basophilic leukemia reporter cell line RS-ATL8. METHODS: Sera from shrimp allergic subjects were subjected to ELISA and Western blots using raw or cooked shrimp extract as antigens. Pooled sera were used to sensitize the RS-ATL8 reporter cell line and cells were activated by shrimp extract. Eluted protein extracts separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were tested on the RS-ATL8 cell line and subjected to mass spectrometry to identify potential candidate allergens. RESULTS: Allergic sera reacted stronger to raw shrimp extract than cooked shrimp extract (P=0.009). Western blot demonstrated that major IgE reactivity protein bands were at 32–39 kDa and 91–230 kDa in both raw and cooked shrimp extracts. The eluted protein bands at the molecular weight of 38 and 115 kDa from raw shrimp extract induced IgE cross-linking as assayed by the RS-ATL8 cell line. These protein bands were subjected to mass spectrometry for analysis. Ubiquitin-activating enzyme and crustacyanin were identified as potential candidate novel shrimp allergens. CONCLUSIONS: The RS-ATL8 reporter cell line can be used to identify potential new shrimp allergens that can functionally cross-link IgE and induce mast cell degranulation.
Allergens ; Animals ; Basophils ; Blotting, Western ; Cell Line ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin E ; Immunoglobulins ; Leukemia ; Mass Spectrometry ; Mast Cells ; Molecular Weight ; Penaeidae ; Rats ; Shellfish Hypersensitivity ; Sodium Dodecyl Sulfate ; Tigers ; Ubiquitin-Activating Enzymes

Extracellular interleukin 1 alpha (IL-1α) released from keratinocytes is one of the endpoints for in vitro assessments of skin irritancy. Although cells dying via primary skin irritation undergo apoptosis as well as necrosis, IL-1α is not released in apoptotic cells. On the other hand, active secretion has been identified in interleukin-1 receptor antagonist (IL-1ra), which was discovered to be a common, upregulated, differentially-expressed gene in a microarray analysis performed with keratinocytes treated using cytotoxic doses of chemicals. This study examined whether and how IL-1ra, particularly extracellularly released IL-1ra, was involved in chemically-induced keratinocyte cytotoxicity and skin irritation. Primary cultured normal adult skin keratinocytes were treated with cytotoxic doses of chemicals (hydroquinone, retinoic acid, sodium lauryl sulfate, or urshiol) with or without recombinant IL-1ra treatment. Mouse skin was administered irritant concentrations of hydroquinone or retinoic acid. IL-1ra (mRNA and/or intracellular/extracellularly released protein) levels increased in the chemically treated cultured keratinocytes with IL-1α and IL-1β mRNAs and in the chemically exposed epidermis of the mouse skin. Recombinant IL-1ra treatment significantly reduced the chemically-induced apoptotic death and intracellular/extracellularly released IL-1α and IL-1β in keratinocytes. Collectively, extracellular IL-1ra released from keratinocytes could be a compensatory mechanism to reduce the chemically-induced keratinocyte apoptosis by antagonism to IL-1α and IL-1β, suggesting potential applications to predict skin irritation.
Adult ; Animals ; Apoptosis* ; Epidermis ; Hand ; Humans ; In Vitro Techniques ; Interleukin 1 Receptor Antagonist Protein ; Interleukin-1* ; Interleukin-1alpha ; Keratinocytes* ; Mice ; Microarray Analysis ; Necrosis ; RNA, Messenger ; Skin ; Sodium Dodecyl Sulfate ; Tretinoin

PURPOSE: The immunological characteristics of young Korean children with walnut (WN) allergy and the influence of different cooking methods on WN proteins have not been evaluated to date. This study aimed to evaluate the major WN allergens identified among Korean children, together with changes in WN antigenicity caused by common cooking methods. METHODS: We enrolled children under the age of 13 years with WN serum-specific immunoglobulin (Ig) E concentrations. The protein fractions of dry-fried and boiled WN extracts were compared with those of raw WNs using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-dimentional gel electrophoresis (2DE) and a proteomic analysis using electrospray ionization (liquid chromatography-mass spectrometry [LC-MS]). An immunoblotting analysis was conducted to examine IgE reactivity toward raw WNs using serum samples from 6 children with a clinical WN allergy. To determine the processed WN proteins with IgE-binding capacity, a 2D-immunoblotting analysis was performed using the pooled sera of 20 WN-sensitized children. RESULTS: Protein bands from raw WNs were identified at 9, 16, 28, 52, 58, and 64 kDa via SDS-PAGE. The 9- and 16-kDa protein bands were enhanced by boiling, whereas the 52- and 64-kDa bands were considerably diminished. On LC-MS analysis, of the 66 IgE-binding proteins present in raw WNs, 57 were found in dry-fried WNs, but only 4 in boiled WNs. The sera of 5 out of 6 participants reacted with the 52-kDa protein bands and those of 4 out of 6 participants reacted with the 16- and 28-kDa protein bands, respectively. Meanwhile, a 2D-immunoblotting result confirmed the presence of different binding patterns among children who consumed cooked WNs. CONCLUSIONS: The protein profile of boiled WNs is substantially different from that of raw WNs. However, 4 proteins including prolamins remained stable after dry-frying or boiling. Further studies are needed to evaluate the clinical relevance of these findings.
Allergens ; Child* ; Cooking ; Electrophoresis ; Electrophoresis, Polyacrylamide Gel ; Humans ; Hypersensitivity ; Immunoblotting ; Immunoglobulin E ; Immunoglobulins* ; Juglans* ; Prolamins ; Sodium Dodecyl Sulfate ; Spectrum Analysis

To develop decellularized heart valve scaffold from porcine for heart valve regeneration. Porcine heart valves were decellularized with unique optimized approach by using 1% sodium dodecyl sulfate solution and 5% dimethyl sulfoxide for the first time. Effect of decellularization process on scaffold were characterized by hematoxylin-eosin, 4′,6-diamidino-2-phenylindole, Masson's trichrome, alcian blue staining and scanning electron microscopy for extracellular matrix (ECM) analysis in scaffold. The results showed that developed protocol for decellularization of heart valve scaffold shown complete removal of all cellular components, without changing the properties of ECM. The developed protocol was successfully used for heart valve ECM scaffolds development from porcine. The developed protocol seems to be promising solution for the heart valve tissue engineering application.
Alcian Blue ; Detergents* ; Dimethyl Sulfoxide ; Extracellular Matrix ; Heart Valves* ; Heart* ; Microscopy, Electron, Scanning ; Regeneration ; Sodium Dodecyl Sulfate ; Tissue Engineering*

PURPOSE: Helicobacter pylori is a widely distributed gram-negative bacterium that infects the human stomach and duodenum. HpaA is a H. pylori-specific lipoprotein that has been shown to be an effective protective antigen against H. pylori infection. HpaA of H. pylori as a vaccine antigen is fully competent for stimulation of immune responses. The aim of this project is cloning, expression, and purification flagellar sheath adhesion of H. pylori in Escherichia coli host by fast protein liquid chromatography (FPLC) as a vaccination target. MATERIALS AND METHODS: The hpaA gene was inserted into pET28a (+) as cloning and expression vectors respectively. The recombinant plasmid (pET-hpaA) was subjected to sequencing other than polymerase chain reaction (PCR) and digestion analysis. Protein expression was induced by adding 1 mM isopropyl-beta-D-thiogalactoside to cultures of E. coli strain BL21 transformed with pET-hpaA. Protein expression assessed with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Protein purification of flagellar sheath adhesion was by FPLC. RESULTS: The restriction endonuclease digestion, PCR amplification analysis showed that the hpaA gene of 730 bp was amplified from H. pylori DNA and sequencing analysis of the pET-hpaA confirmed the cloning accuracy and in frame insertion of hpaA fragment. SDS-PAGE analysis showed the expression of an approximately 29,000 Da protein. CONCLUSION: Sequencing results along with SDS-PAGE analysis confirms the expression of recombinant hpaA in the heterologous E. coli BL21. Conclusion A prokaryotic expression system for hpaA gene was successfully constructed. These results indicate that production of a specific recombinant protein is an alternative and potentially more expeditious strategy for development of H. pylori vaccine.
Chromatography, Liquid ; Clone Cells* ; Cloning, Organism* ; Digestion ; DNA ; DNA Restriction Enzymes ; Duodenum ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli* ; Escherichia* ; Helicobacter pylori* ; Helicobacter* ; Humans ; Lipoproteins ; Plasmids ; Polymerase Chain Reaction ; Sodium Dodecyl Sulfate ; Stomach ; Vaccination*

BACKGROUND: Grass pollen grains are important causes of respiratory allergies. The Philippines has a different grass flora compared to that of western countries, so pollen extracts have to be processed for use in the diagnosis and treatment of respiratory allergies. The local pollen extracts available in clinical practice have not yet been characterized, which is important in improving extract quality.
OBJECTIVE: This study aims to perform physicochemical characterization through protein content determination and gradient sodium-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of extracts from four grasses: Cynodon dactylon (bermuda grass), Axonopus compressus (carabao grass), Imperata cylindrica (cogon), and Saccharum spontaneum (talahib) and immunologic characterization by identifying its IgE-binding component through immunoblot.
METHODS: This is a descriptive study. The pollen grains were processed into allergen extracts and protein contents were determined. The extracts were separated by gradient SDS-PAGE and subjected to immunoblotting. Bands were visualized using Fluorchem C2 aided with Alpha View Software.
RESULTS: Total protein in the pollen extracts ranged from 281.3-968.61 µg/ml. Protein bands of bermuda were in the 14.4-66.3 kDa range, carabao grass at 3.5-66.3 kDa, cogon at 3.5-200 kDA, and talahib at 21.5-66.3 kDa. A single IgE-binding protein band was seen on immunoblot at 55.4 kDa using a single serum sample.
CONCLUSION: Protein contents of the allergen extracts vary. The molecular weights of the different protein bands seem to correspond to known groups of grass pollen allergens. There was only one IgE-binding protein band seen on preliminary immunoblot.

Allergens ; Bermuda ; Cynodon ; Electrophoresis, Polyacrylamide Gel ; Galectin 3 ; Immunoblotting ; Immunoglobulin E ; Molecular Weight ; Philippines ; Poaceae ; Pollen ; Respiratory Hypersensitivity ; Saccharum ; Sodium ; Sodium Dodecyl Sulfate

PURPOSE: The common triple therapy for Helicobacter pylori is challenged by the increasing cases of antibiotic resistant infections, raising the need to explore alternative therapies. Oral administration of egg yolk immunoglobulin Y (IgY) has been previously reported as a means of passive immunization therapy for H. pylori infections. In this work, we investigated the inhibitory effect of IgY on the attachment of H. pylori to AGS cell line. MATERIALS AND METHODS: Recombinant OipA was prepared. Hens were immunized with recombinant protein three times. IgY was purified from egg yolks of immunized hens using polyethylene glycol precipitation method. The inhibitory effect of the specific immunoglobulin was evaluated in AGS cell line infected with H. pylori. RESULTS: The presence of recombinant OipA (30 kD) was confirmed via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Immunization of hens was confirmed using enzyme-linked immunosorbent assay. The purified IgY from egg yolks were assessed using SDS-PAGE and confirmed by western blot. CONCLUSION: The results showed that IgY-OipA had inhibitory effect on attachment of H. pylori to AGS cell line and may be utilized as a therapeutic or prophylaxis material.
Administration, Oral ; Blotting, Western ; Cell Line* ; Complementary Therapies ; Egg Yolk ; Electrophoresis, Polyacrylamide Gel ; Enzyme-Linked Immunosorbent Assay ; Helicobacter pylori* ; Helicobacter* ; Immunization ; Immunization, Passive ; Immunoglobulins ; Polyethylene Glycols ; Sodium Dodecyl Sulfate

Bee pollen is pollen granules packed by honey bees and is widely consumed as natural healthy supplements. Bee pollen-induced anaphylaxis has rarely been reported, and its allergenic components have never been studied. A 40-year-old male came to the emergency room with generalized urticaria, facial edema, dyspnea, nausea, vomiting, abdominal pain, and diarrhea 1 hour after ingesting one tablespoon of bee pollen. Oxygen saturation was 91%. His symptoms resolved after injection of epinephrine, chlorpheniramine, and dexamethasone. He had seasonal allergic rhinitis in autumn. Microscopic examination of the bee pollen revealed Japanese hop, chrysanthemum, ragweed, and dandelion pollens. Skin-prick with bee pollen extracts showed positive reactions at 0.1 mg/mL (A/H ratio > 3+). Serum specific IgE to ragweed was 25.2, chrysanthemum 20.6, and dandelion 11.4 kU/L; however, Japanese hop, honey-bee venom and yellow-jacket venom were negative (UniCAP(R), Thermo Fisher Scientific, Uppsala, Sweden). Enzyme-linked immunosorbent assay (ELISA) confirmed serum specific IgE to bee-pollen extracts, and an ELISA inhibition assay for evaluation of cross-allergenicity of bee pollen and other weed pollens showed more than 90% of inhibition with chrysanthemum and dandelion and ~40% inhibition with ragweed at a concentration of 1 microg/mL. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and IgE-immunoblot analysis revealed 9 protein bands (11, 14, 17, 28, 34, 45, 52, 72, and 90 kDa) and strong IgE binding at 28-34 kDa, 45 and 52 kDa. In conclusion, healthcare providers should be aware of the potential risk of severe allergic reactions upon ingestion of bee pollen, especially in patients with pollen allergy.
Abdominal Pain ; Adult ; Ambrosia ; Anaphylaxis* ; Asian Continental Ancestry Group ; Bees* ; Chlorpheniramine ; Chrysanthemum ; Dexamethasone ; Diarrhea ; Dyspnea ; Eating ; Edema ; Electrophoresis, Polyacrylamide Gel ; Emergency Service, Hospital ; Enzyme-Linked Immunosorbent Assay ; Epinephrine ; Health Personnel ; Honey ; Humans ; Humulus ; Hypersensitivity ; Immunoglobulin E ; Male ; Nausea ; Oxygen ; Pollen ; Rhinitis, Allergic, Seasonal ; Sodium Dodecyl Sulfate ; Taraxacum ; Urticaria ; Venoms ; Vomiting

PURPOSE: This study aimed to investigate the efficacy of cleaning solutions on saliva-contaminated zirconia in comparison to air-abrasion in terms of resin bonding. MATERIALS AND METHODS: For saliva-contaminated airabraded zirconia, seven cleaning methods)-no contamination (NC), water-spray rinsing (WS), additional airabrasion (AA), and cleaning with four solutions (Ivoclean [IC]; 1.0 wt% sodium dodecyl sulfate [SDS], 1.0 wt% hydrogen peroxide [HP], and 1.0 wt% sodium hypochlorite [SHC])-were tested. The zirconia surfaces for each group were characterized using various analytical techniques. Three bonded resin (Panavia F 2.0) cylinders (bonding area: 4.5 mm2) were made on one zirconia disk specimen using the Ultradent jig method [four disks (12 cylinders)/group; a total of 28 disks]. After 5,000 thermocycling, all specimens were subjected to a shear bond strength test with a crosshead speed of 1.0 mm/minute. The fractured surfaces were observed using an optical and scanning electron microscope (SEM). RESULTS: Contact angle measurements showed that groups NC, AA, IC, and SHC had hydrophilic surfaces. The X-ray photoelectron spectroscopy (XPS) analysis showed similar elemental distributions between group AA and groups IC and SHC. Groups IC and SHC showed statistically similar bond strengths to groups NC and AA (P>.05), but not groups SDS and HP (P<.05). For groups WS, SDS, and HP, blister-like bubble formations were observed on the surfaces under SEM. CONCLUSION: Within the limitations of this in vitro study, some of the cleaning solutions (IC or SHC) were effective in removing saliva contamination and enhancing the resin bond strength.
Dental Bonding ; Hydrogen Peroxide ; Photoelectron Spectroscopy ; Saliva ; Sodium Dodecyl Sulfate ; Sodium Hypochlorite