BACKGROUND: Flow cytometric crossmatching (FCXM) is widely used in hospitals performing solid organ transplantation. Pronase treatment of lymphocytes can increase the sensitivity and specificity of B-cell FCXM. However, it can also affect human leukocyte antigen (HLA) expression and results of FCXM. We treated lymphocytes with various concentrations of pronase and analysed the effect of the treatment on the FCXM results. METHODS: The peripheral blood mononuclear cells isolated from 10 renal transplant donors were treated with three different concentrations of pronase (0.5, 1.0, and 2.0 mg/mL). The effects of pronase on median fluorescence intensity (MFI) values of AB serum (Fcγ receptor), HLA class I and II, and on the MFI ratio of HLA class I and II were analysed. RESULTS: In B-cell FCXM, the MFI values of AB serum (Fcγ receptor) and HLA class I were significantly decreased by the pronase treatment. The MFI ratio of HLA class II was significantly increased upon treatment with 0.5, 1.0, and 2.0 mg/mL pronase (P<0.05, P<0.01, and P<0.01, respectively). In T-cell FCXM, the MFI ratio of HLA class I was significantly decreased by the pronase treatment (all P<0.01). CONCLUSIONS: When performing FCXM, it is recommended that B-lymphocytes should be treated with 1.0 or 2.0 mg/mL pronase. In the case of T-lymphocytes, pronase treatment should be adopted with caution.
B-Lymphocytes ; Flow Cytometry ; Fluorescence ; Humans ; Leukocytes ; Lymphocytes ; Organ Transplantation ; Pronase* ; Sensitivity and Specificity ; T-Lymphocytes ; Tissue Donors ; Transplants

BACKGROUND/AIMS: Helicobacter pylori colonizes on the apical surface of gastric surface mucosal cells and the surface mucous gel layer. Pronase is a premedication enzyme for endoscopy that can disrupt the gastric mucus layer. We evaluated the additive effects of pronase combined with standard triple therapy for H. pylori eradication. METHODS: This prospective, single-blinded, randomized, controlled study was conducted between June and October 2012. A total of 116 patients with H. pylori infection were enrolled in the study (n=112 patients, excluding four patients who failed to meet the inclusion criteria) and were assigned to receive either the standard triple therapy, which consists of a proton pump inhibitor with amoxicillin and clarithromycin twice a day for 7 days (PAC), or pronase (20,000 tyrosine units) combined with the standard triple therapy twice a day for 7 days (PACE). RESULTS: In the intention-to-treat analysis, the eradication rates of PAC versus PACE were 76.4% versus 56.1% (p=0.029). In the per-protocol analysis, the eradication rates were 87.5% versus 68.1% (p=0.027). There were no significant differences concerning adverse reactions between the two groups. CONCLUSIONS: According to the interim analysis of the trial, pronase does not have an additive effect on the eradication of H. pylori infection (ClinicalTrial.gov: NCT01645761).
Adult ; Amoxicillin/therapeutic use ; Anti-Bacterial Agents/*therapeutic use ; Clarithromycin/therapeutic use ; Drug Administration Schedule ; Drug Therapy, Combination/methods ; Female ; Gastric Mucosa/drug effects/microbiology ; Helicobacter Infections/*drug therapy ; Helicobacter pylori/*drug effects ; Humans ; Male ; Middle Aged ; Pronase/*therapeutic use ; Prospective Studies ; Proton Pump Inhibitors/*therapeutic use ; Single-Blind Method ; Treatment Outcome

No abstract available.
Anti-Bacterial Agents/*therapeutic use ; Female ; Helicobacter Infections/*drug therapy ; Helicobacter pylori/*drug effects ; Humans ; Male ; Pronase/*therapeutic use ; Proton Pump Inhibitors/*therapeutic use

BACKGROUND AND OBJECTIVES: Glutaraldehyde (GA) has been used as a representative method of tissue preservation in cardiovascular surgery. However, GA has showed limited durability including calcification, mechanical failure and toxicity. To overcome those unsolved problems, we analyzed the crosslinking differences of primary amines, GA and genipin in their mechanical and biochemical properties with a single or double crosslinking agent for clinical application. MATERIALS AND METHODS: Samples were divided into 3 groups; control, single crosslinking fixation and double crosslinking fixation after decellurarization using bovine pericardium. For analysis of the biochemical and mechanical properties of each crosslinking method, tensile strength, percentage strain, thermal stability, resistance to pronase, nynhydrin and cytotoxicity test were studied. RESULTS: Combined hexamethylene diamine and suberic acid in the carbodiimide hydrochloride/N-hydroxysucinimide solution (EDC/NHS) after decellurarization, tensile strength and strain percentage were not statistically significant compared to the single crosslinking treated groups (p>0.05). Tissue crosslinking stability was weak in single treatment of diphenylphosphoryl azide, suberic acid, low concentration of EDC, hexamethylene diamine and procyanidin groups, but thermal stability and resistance to the pronase and ninhydrin were markedly increased in concentrated EDC/NHS or after combined double treatment with low concentration of GA or genipin (p<0.001). CONCLUSION: Single or double crosslinking with low concentration of carbodiimide, diphenylphosphonyl azide, procyanidin, suberic acid and hexane diamine were not as effective in mechanical, biochemical, cytotoxic and crosslinking properties compared to GA or genipin fixation, but their mechanical and chemical properties were much improved when combined with low concentrations of GA or genipin in the double crosslinking process.
Amines ; Azides ; Biflavonoids ; Bioprosthesis ; Caprylates ; Catechin ; Dicarboxylic Acids ; Glutaral ; Iridoids ; Ninhydrin ; Pericardium ; Proanthocyanidins ; Pronase ; Sprains and Strains ; Tensile Strength ; Tissue Preservation

BACKGROUND/AIMS: Gastric mucus should be removed before endoscopic examination to increase visibility. In this study, the effectiveness of premedication with pronase for improving visibility during endoscopy was investigated. METHODS: From April 2010 to February 2011, 400 outpatients were randomly assigned to receive endoscopy with one of four premedications as follows: dimethylpolysiloxane (DMPS), pronase and sodium bicarbonate with 10 minutes premedication time (group A, n=100), DMPS and sodium bicarbonate with 10 minutes premedication time (group B, n=100), DMPS, pronase and sodium bicarbonate with 20 minutes premedication time (group C, n=100), and DMPS and sodium bicarbonate with 20 minute premedication time (group D, n=100). One endoscopist, who was unaware of the premedication types, calculated the visibility scores (range, 1 to 3) of the antrum, lower gastric body, upper gastric body and fundus. The sum of the scores from the four locations was defined as the total visibility score. RESULTS: Group C showed significantly lower scores than other groups (p=0.002). Group C also had the lowest frequency of flushing, which was significantly lower than that of group D. Groups C and D had significantly shorter durations of examination than groups A and B. CONCLUSIONS: Using pronase 20 minutes before endoscopy significantly improved endoscopic visualization and decreased the frequency of water flushing.
Dimethylpolysiloxanes ; Endoscopy ; Flushing ; Humans ; Mucous Membrane ; Mucus ; Outpatients ; Premedication ; Pronase ; Sodium Bicarbonate ; Unithiol

Since most of the respiratory epithelial cell lines are descended from neoplastic tissues or have been fused with neoplastic cells when being selected to a cell line, their biological behaviors are far different from normal respiratory epithelial cells. Aiming at better reflecting the biological properties of epithelial cells under respiratory pathological conditions, our study probed into a new isolation technique and culture method of mice respiratory epithelial cell. Pronase was applied for cell isolation from mouse trachea, and a modified medium with collagen-coated plate for primary culture. The ciliary movement can be observed under microscope, which demonstrates that the original biological functions of the tracheal epithelial cell have been reserved with our method. The research presents the efficacy, convenience and reliability of the separation technique and culture method established by this study, and laying preferable condition for cell models of respiratory diseases. Meanwhile, this method may be used for other animal models.
Animals ; Cell Separation ; methods ; Culture Media ; Epithelial Cells ; cytology ; Female ; Male ; Mice ; Mice, Inbred BALB C ; Primary Cell Culture ; methods ; Pronase ; pharmacology ; Trachea ; cytology

This study was designed to investigate the effects an 8-Br-cGMP on the neuronal activity of rat vestibular nuclear cells. Sprague-Dawley rats aged 14 to 16 days were decapitated under ether anesthesia. After treatment with pronase and thermolysin, the dissociated vestibular nuclear cells were transferred into a chamber on an inverted microscope. Spontaneous action potentials and potassium currents were recorded by standard patch-clamp techniques under current and voltage-clamp modes. Twelve vestibular nuclear cells revealed excitatory responses to 1-5 microM of 8-Br-cGMP, and 3 neurons did not respond to 8-Br-cGMP. Whole potassium currents of vestibular nuclear cells were decreased by 8-Br-cGMP (n=12). After calcium-dependent potassium currents were blocked by tetraethylammonium, the potassium currents were not decreased by 8-Br-cGMP. These experimental results suggest that 8-Br-cGMP changes the neuronal activity of vestibular nuclear cells by blocking the calcium-dependent potassium currents that underlie the afterhyperpolarization.
Action Potentials ; Aged ; Anesthesia ; Animals ; Ether, Ethyl ; Humans ; Neurons ; Nucleotides, Cyclic ; Patch-Clamp Techniques ; Potassium ; Pronase ; Rats ; Rats, Sprague-Dawley ; Tetraethylammonium ; Thermolysin

BACKGROUND: Effective decellularization and fixation process is critical, in order to use xenogenic valves clinically. In the present study, we decellularized bovine pericardium using sodium dodecyl sulfate (SDS) and N-lauroyl sarcosinate, treated with alpha-galactosidase, and then fixed in various manners, to find out the most effective tissue preservation & fixation procedure. MATERIAL AND METHOD: Bovine pericardium was decellularized with SDS and N-lauroyl sarcosinate, and treated with alpha-galactosidase. Both groups were fixed differently, by varying glutaraldehyde (GA) or EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide)/N-hydroxysuccinamide (NHS) treatment conditions. Thereafter, physical examination, tensile strength test, thermal stability test, cytotoxicity test, pronase test, pronase-ninhydrin test, purpald test, permeability test, compliance test, H&E staining, DNA quantification, and alpha-galactose staining were carried out to each groups. RESULT: GA fixed groups showed better physical properties and thermal stability than EDC/NHS fixed groups. EDC/NHS-GA dual fixed groups showed better physical properties and thermal stability than EDC/NHS fixed groups, and showed better thermal stability than GA fixed groups. In pronase test and pronase-ninhydrin test, GA fixed groups and EDC/NHS-GA dual fixed groups showed stronger crosslinks than EDC/NHS groups. Permeability and compliance tended to increase in EDC/NHS-GA dual fixed groups, compared to GA fixed groups. But, EDC/NHS-GA dual fixed groups had stronger tensile strength and lower cytotoxicity than GA fixed groups. CONCLUSION: We have verified that EDC/NHS-GA dual fixation can make effective crosslinks and lower the toxicity of GA fixation. Henceforth, we will verify if EDC/NHS-GA dual fixation can lower calcifications & tissue failure in vivo experiment.
alpha-Galactosidase ; Compliance ; DNA ; Glutaral ; Pericardium ; Permeability ; Physical Examination ; Pronase ; Sodium Dodecyl Sulfate ; Sulfhydryl Compounds ; Tensile Strength ; Tissue Engineering ; Tissue Preservation ; Transplantation, Heterologous

OBJECTIVE: The aim of this study is to elucidate the effects of transforming growth factor-beta (TGF-beta)1 and L-ascorbic acid on proteoglycan synthesis, and the relationship between Sox9, proteoglycan, and TGF-beta1 in intervertebral disc cells. METHODS: Human intervertebral disc tissue was sequentially digested to 0.2% pronase and 0.025% collagenase in DMEM/F-12 media and extracted cells were cultured in 37degrees C, 5% CO2 incubator. When intervertebral disc cells were cultured with TGF-beta1 or L-ascorbic acid, the production level of sulfated glycosaminoglycan (sGAG) was estimated by dimethyl methyleneblue (DMMB) assay. The changes of Sox9 mRNA and protein levels via TGF-beta1 were detected by RT-PCR and Western blot analysis in each. RESULTS: The amount of sGAG was increased with the lapse of time during incubation, and sGAG content of pellet cultured cells was much larger than monolayer culture. When primary cultured intervertebral disc cells in monolayer and pellet cultures were treated by TGF-beta1 20 ng, sGAG content of experimental group was increased significantly compared to control group in both cultures. L-Ascorbic acid of serial concentrations (50-300 ug/ml) increased sGAG content of mono layer cultured intervertebral disc cells significantly in statistics. The co-treatment of TGF-beta1 and L-ascorbic acid increased more sGAG production than respective treatment. After treating with TGF-beta1, Sox9 mRNA and protein expression rates were significantly increased in disc cells compared with the control group. CONCLUSION: This study suggests that TGF-beta1 would increase sulfated glycosaminoglycan (sGAG) and other proteoglycans such as versican by elevating Sox9 mRNA and protein expressions in order.
Ascorbic Acid ; Blotting, Western ; Cells, Cultured ; Collagenases ; Glycosaminoglycans ; Humans ; Incubators ; Intervertebral Disc ; Pronase ; Proteoglycans ; RNA, Messenger ; Transforming Growth Factor beta1 ; Versicans

We evaluated the ability of lactic acid bacteria, Weissella cibaria, isolated from the oral cavity to adhere to epithelial cells. W. cibaria efficiently adhered to KB cells and HeLa cells. In addition, W. cibaria efficiently adhered to Fusobacterium nucleatum. But the adhesiveness of W. cibaria disappeared upon exposure to LiCl or pronase, suggesting that the S-layer proteins of W. cibaria mediated the adhesiveness. The molecular mass of the S-layer proteins extracted from W. cibaria was approximately 50 kDa. When W. cibaria strains were washed with 0.45% saline, the bacteria were efficiently adhered to the epithelial cells. In conclusion, W. cibaria has the ability to adhere to epithelial cells through the S-layer proteins.
Adhesiveness ; Bacteria ; Epithelial Cells* ; Fusobacterium nucleatum ; HeLa Cells ; Humans ; KB Cells ; Lactic Acid ; Mouth ; Pronase ; Weissella*