Pre-procedure oral administration of pronase improves efficacy of lugol chromoendoscopy in esophageal squamous cell carcinoma screening: a prospective, double-blinded, randomized, controlled trial.

BACKGROUND AND AIMS: Chromoendoscopy with Lugol's staining is used to screen for early esophageal squamous cell carcinoma (ESCC). Its efficacy is greatly limited by unstandardized defoaming preparation. This study aimed to confirm whether pre-procedure oral administration of pronase could improve the diagnostic performance of Lugol chromoendoscopy in high-risk patients being screened for early ESCC. METHODS: A total of 955 patients at-risk were prospectively recruited for screening for ESCC. Patients were randomly assigned (1:1) to groups with or without (control group) pronase administration. Endoscopic diagnosis of early ESCC was based on the presence of pink-color sign in Lugol's unstained area, and a biopsy was routinely conducted if the Lugol's unstained lesion was larger than 0.5 cm. The early cancer detection rate was used as the primary endpoint. RESULTS: Pre-procedure oral administration of pronase improved mucosal visibility during Lugol chromoendoscopy (P = 0.008). There were no differences in the number of Lugol's unstained lesions between the 2 groups (23.27% [111/477] vs. 25.11% [120/478], P = 0.508). Meaningfully, the detection rate of ESCC (confirmed by histopathology) was significantly higher in the pronase group than in the control group (27.03% [30/111] vs. 17.50% [21/120], P = 0.041), as well as the detection rate of lesions with pink-color sign during chromoendoscopy (35.14% [39/111] vs. 13.33% [16/120], P < 0.001). The diagnostic performance of Lugol chromoendoscopy had improved with the use of pronase (area under the curve = 0.85 vs. 0.69, P = 0.019), accompanied by an increased sensitivity (86.67% vs. 47.62%, P = 0.004). There was no difference in the adverse events between the 2 groups (P = 0.793). CONCLUSIONS: Pre-procedure oral administration of pronase significantly increased the detection rate of early ESCC and optimized the diagnostic performance of Lugol chromoendoscopy, which should be recommended during routine endoscopic screening for early ESCC in high-risk patients. TRIAL REGISTRATION: Pronase improves efficacy of Lugol chromoendoscopy screening on esophageal cancerous lesions (NCT02030769).

Insufficiency of collagenases in establishment of primary chondrocyte culture from cartilage of elderly patients receiving total joint replacement.

Background Collagenases are frequently used in chondrocyte isolation from articular cartilage. However, the sufficiency of this enzyme in establishing primary human chondrocyte culture remains unknown. Methods Cartilage slices shaved from femoral head or tibial plateau of patients receiving total joint replacement surgery (16 hips, 8 knees) were subjected to 0.02% collagenase IA digestion for 16 h with (N = 19) or without (N = 5) the pre-treatment of 0.4% pronase E for 1.5 h. Chondrocyte yield and viability were compared between two groups. Chondrocyte phenotype was determined by the expression ratio of collagen type II to I. The morphology of cultured chondrocytes was monitored with a light microscope.Results Cartilage with pronase E pre-treatment yielded significantly higher chondrocytes than that without the pre-treatment (3,399 ± 1,637 cells/mg wet cartilage vs. 1,895 ± 688 cells/mg wet cartilage; P = 0.0067). Cell viability in the former group was also significantly higher than that in the latter (94% ± 2% vs. 86% ± 6%; P = 0.03). When cultured in monolayers, cells from cartilage with pronase E pre-treatment grew in a single plane showing rounded shape while cells from the other group grew in multi-planes and exhibited irregular shape. The mRNA expression ratio of collagen type II to I was 13.2 ± 7.5 in cells isolated from cartilage pre-treated with pronase E, indicating a typical chondrocyte phenotype. Conclusions Collagenase IA was not sufficient in establishing primary human chondrocyte culture. Cartilage must be treated with pronase E prior to collagenase IA application.

DNAJB9-positive monotypic fibrillary glomerulonephritis is not associated with monoclonal gammopathy in the vast majority of patients.

The association of fibrillary glomerulonephritis (FGN) with monoclonal gammopathy has been controversial, although monotypic FGN is currently classified as a monoclonal gammopathy of renal significance (MGRS) lesion. To define this lesion, we correlated findings by immunofluorescence on frozen and paraffin tissue, IgG subtype staining and serum protein electrophoresis with immunofixation in patients with monotypic FGN. Immunofluorescence was performed on paraffin sections from 35 cases of DNAJB9-associated FGN that showed apparent light chain restriction of glomerular IgG deposits by standard immunofluorescence on frozen tissue. On paraffin immunofluorescence, 15 cases (14 lambda and one kappa restricted cases on frozen tissue immunofluorescence) showed no light chain restriction, 19 showed similar light chain restriction, and one was negative for both light chains. Seven of the 15 cases with masked polyclonal deposits also had IgG subclass restriction and these cases would have been diagnosed as a form of monoclonal protein-associated glomerulonephritis if paraffin immunofluorescence was not performed. Monotypic FGN (confirmed by paraffin immunofluorescence and IgG subclass restriction) accounted for only one of 151 (0.7%) patients with FGN encountered during the last two years. Only one of 11 of cases had a detectable circulating monoclonal protein on serum protein electrophoresis with immunofixation. We propose that paraffin immunofluorescence is required to make the diagnosis of lambda-restricted monotypic FGN as it unmasked polytypic deposits in over half of patients. When confirmed by paraffin immunofluorescence and IgG subclass staining, DNAJB9-positive monotypic FGN is very rare and is not associated with monoclonal gammopathy in the vast majority of patients. Thus, there is a question whether this lesion should be included in MGRS-related diseases.

Effect of premedication on lesion detection rate and visualization of the mucosa during upper gastrointestinal endoscopy: a multicenter large sample randomized controlled double-blind study.

BACKGROUND: Premedication in upper gastrointestinal endoscopy for higher lesions detection rate has not been well studied so far. This study aimed to confirm whether premedication could improve the detection rate of early cancer or precancerous lesions and mucosal visibility. METHOD: From July 2015 to December 2015, 7200 participants from 6 centers were screened by endoscopy with one of the 4 following premedications randomly: (1) water (group D); (2) pronase (group A); (3) simethicone (group B); (4) pronase and simethicone (group C). Early cancer and precancerous lesions detection rates were taken as the primary endpoints, and mucosal visibility was taken as the secondary endpoint. They were compared among four groups to determine different premedication effects in terms of different anatomical sites. Trial was registered at Chinese Clinical Trial Registry; the registration number is ChiCTR-IOR-17010985. RESULTS: The upper gastrointestinal overall precancerous lesion detection rates among four groups were 8.7, 8.4, 10.0, and 10.3%, the overall early cancer detection rates were 1.3, 1.4%, 1.5, and 1.6%, both without significant difference (p = 0.138 and 0.878). However, the visibility score distributions between control group (D) and premedication groups (A, B, and C) were all statistically significant, with all anatomical sites p values < 0.001. Subgroup analyses, from 2 centers without screening before, also showed significant difference in esophageal (3.9, 3.3, 4.5, and 8.4% with p = 0.004) and overall (7.0, 5.5, 7.3, and 12.0% with p = 0.004) precancerous lesion detection rate. CONCLUSIONS: Premedication with pronase and simethicone may not increase lesion detection rates but could significantly increase the upper gastrointestinal mucosal visibility.

Efficacy and safety of using premedication with simethicone/Pronase during upper gastrointestinal endoscopy examination with sedation: A single center, prospective, single blinded, randomized controlled trial.

BACKGROUND AND AIM: To investigate the efficacy and safety of premedication with simethicone/Pronase during esophagogastroduodenoscopy (EGD) with sedation. METHODS: Six hundred and ten patients were randomly allocated to two groups based on type of premedication given. Premedication used in the control group was 10 mL lidocaine hydrochloride mucilage (LHM, N = 314) and premedication used in the intervention group was 80 mL simethicone/Pronase solution plus 10 mL lidocaine hydrochloride mucilage (SP/LHM, N = 296). EGD was done under sedation. Visibility scores, number of mucosal areas that needed cleansing, water consumption for cleansing, time taken for examination, diminutive lesions, pathological diagnosis, patients' gag reflex and oxygenation (pulse oximetry) were recorded. RESULTS: SP/LHM has significantly lower total visibility score than LHM (7.978 ± 1.526 vs 6.348 ± 1.097, P < 0.01). During the procedure, number of intragastric areas that needed cleansing and amount of water consumed were significantly less in the SP/LHM than in the LHM group (P < 0.01). In SP/LHM (P = 0.01), endoscopy procedure duration was significantly longer. Although there was no significant difference in rate of detection of diminutive lesions between LHM and SP/LHM, the endoscopist carried out more biopsies in SP/LHM. This led to a higher rate of diagnosis of atrophic gastritis (P = 0.014) and intestinal metaplasia (P = 0.024). There was no significant difference in gag reflex (P = 0.604) and oxygenation during the endoscopy procedure for either group of patients. CONCLUSION: Routine use of premedication with simethicone/Pronase should be recommended during EGD with sedation.

Ephestia kuehniella tolerance to Bacillus thuringiensis Cry1Aa is associated with reduced oligomer formation.

The basis of the different susceptibility of Ephestia kuehniella to the Cry1Aa and Cry1Ac δ-endotoxins from Bacillus thuringiensis kurstaki BNS3 was studied. Both toxins bound specifically to the BBMV of E. kuehniella. The result of the ligand blot showed that Cry1Ac bound to three putative receptors of about 100, 65 and 80 kDa and Cry1Aa interacted only with a 100 kDa protein. Pronase digestion of the BBMV-bound toxins was used to analyze the toxin insertion. Both toxins inserted into the BBMV as monomers however, a 14 kDa peptide of α4-α5 which correspond to the oligomeric form of this peptide was detected in case of Cry1Ac only. Analysis of the in vitro oligomerisation of these toxins in the presence of the BBMV of E. kuehniella showed reduced oligomer formation in case of Cry1Aa in comparison with Cry1Ac. Taken together, these results strongly suggest that the difference of toxicity between Cry1Aa and Cry1Ac to E. kuehniella is due to a deficient oligomerisation of Cry1Aa.

Quantifying the combined effects of pronase and benzalkonium chloride in removing late-stage Listeria monocytogenes-Escherichia coli dual-species biofilms.

This work presents the assessment of the effectivity of a pronase (PRN)-benzalkonium chloride (BAC) sequential treatment in removing Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel (SS) using fluorescence microscopy and plate count assays. The effects of PRN-BAC on the occupied area (OA) by undamaged cells in 168 h dual-species samples were determined using a first-order factorial design. Empirical equations significantly (r2 = 0.927) described a negative individual effect of BAC and a negative interactive effect of PRN-BAC achieving OA reductions up to 46%. After treatment, high numbers of remaining attached and released viable and cultivable E. coli cells were detected in PRN-BAC combinations when low BAC concentrations were used. Therefore, at appropriate BAC doses, in addition to biofilm removal, sequential application of PRN and BAC represents an appealing strategy for pathogen control on SS surfaces while hindering the dispersion of live cells into the environment.

Tolerance development in Listeria monocytogenes-Escherichia coli dual-species biofilms after sublethal exposures to pronase-benzalkonium chloride combined treatments.

This study was designed to assess the effects that sublethal exposures to pronase (PRN) and benzalkonium chloride (BAC) combined treatments have on Listeria monocytogenes-Escherichia coli dual-species biofilms grown on stainless steel in terms of tolerance development (TD) to these compounds. Additionally, fluorescence microscopy was used to observe the changes of the biofilm structure. PRN-BAC exposure was carried out using three different approaches and TD was evaluated treating biofilms with a final 100 µg/ml PRN followed by 50 µg/ml BAC combined treatment. Results showed that exposure to PRN-BAC significantly decreased the number of adhered L. monocytogenes (P < 0.05), while E. coli counts remained generally unaltered. It was also demonstrated that the incorporation of recovery periods during sublethal exposures increased the tolerance of both species of the mixed biofilm to the final PRN-BAC treatment. Moreover, control biofilms became more resistant to PRN-BAC if longer incubation periods were used. Regardless of the treatment used, log reduction values were generally lower in L. monocytogenes compared to E. coli. Additionally, microscopy images showed an altered morphology produced by sublethal PRN-BAC in exposed L. monocytogenes-E. coli dual-species biofilms compared to control samples. Results also demonstrated that L. monocytogenes-E. coli dual-species biofilms are able to develop tolerance to PRN-BAC combined treatments depending on way they have been previously exposed. Moreover, they suggest that the generation of bacterial tolerance should be included as a parameter for sanitation procedures design.

Effects of Various Concentrations of Pronase on Flow Cytometric Crossmatching Patients Treated With Rituximab and Donor HLA-Specific Antibodies.

Background: Pronase pretreatment can reduce rituximab (RTX) interference by degrading CD20 in B-cell flow cytometry crossmatch (FCXM) testing. However, it may also reduce the assay sensitivity by degrading HLA molecules. We investigated the effects of various pronase concentrations on RTX interference and the analytical sensitivity of B-cell FCXM testing. Methods: Using 59 patient serum samples and 38 donor lymphocyte samples, we designed 97 recipient-donor pairs and divided them into three groups according to RTX use and the presence of weak-to-moderate donor HLA-specific antibody (DSA) reactions: RTX+/DSA-, RTX+/DSA+, and RTX-/DSA+. FCXM was performed after pretreating lymphocytes with six different pronase concentrations (0, 0.5, 1, 2, 3, and 4 mg/mL). Results: With B-FCXM testing, false-positive results due to RTX in the RTX+/DSA- group markedly decreased with increasing pronase concentrations. The median channel shift values in the RTX+/DSA+ and RTX-/DSA+ groups did not significantly decrease when the pronase concentration was increased from 1 mg/mL to 2 or 3 mg/mL. All eight RTX+/DSA+ cases that were positive at 1 mg/mL pronase but negative at 2 or 3 mg/mL had mean fluorescence intensity (MFI) DSA values of less than 3,000 except for DQ5 (MFI: 5,226). With T-cell FCXM, false-positive results were observed in 2.9% of 315 FCXM tests with pronase pretreatment. Conclusions: Higher concentrations (2 or 3 mg/mL) of pronase effectively eliminated RTX interference but still carried a risk for false negativity for weak DSA reactions in B-cell FCXM. Higher pronase concentrations can be used as an auxiliary method to detect moderate-to-strong DSA reactions in RTX-treated patients.

Comparison of Pronase versus Manual Dechorionation of Zebrafish Embryos for Small Molecule Treatments.

Zebrafish are a powerful animal model for small molecule screening. Small molecule treatments of zebrafish embryos usually require that the chorion, an acellular envelope enclosing the embryo, is removed in order for chemical compounds to access the embryo from the bath medium. For large-scale studies requiring hundreds of embryos, manual dechorionation, using forceps, can be a time-consuming and limiting process. Pronase is a non-specific protease that is widely used as an enzymatic alternative for dechorionating zebrafish embryos. However, whether pronase treatments alter the effects of subsequent small molecule treatments has not been addressed. Here, we provide a detailed protocol for large-scale pronase dechorionation of zebrafish embryos. We tested whether pronase treatment can influence the efficacy of drug treatments in zebrafish embryos. We used a zebrafish model for Duchenne muscular dystrophy (DMD) to investigate whether the efficacies of trichostatin-A (TSA) or salermide + oxamflatin, small molecule inhibitors known to ameliorate the zebrafish dmd muscle degeneration phenotype, are significantly altered when embryos are treated with pronase versus manual dechorionation. We also tested the effects of pronase on the ability of the anthracycline cancer drug doxorubicin to induce cardiotoxicity in zebrafish embryos. When comparing pronase- versus forceps-dechorionated embryos used in these small molecule treatments, we found no appreciable effects of pronase on animal survival or on the effects of the small molecules. The significant difference that was detected was a small improvement in the ability of salermide + oxamflatin to ameliorate the dmd phenotype in pronase-treated embryos when compared with manual dechorionation. Our study supports the use of pronase treatment as a dechorionation method for zebrafish drug screening experiments.

Stable isotope labeling differential glycans discovery in the serum of acute myocardial infarction by ultrahigh-performance liquid chromatography-quadrupole-Orbitrap high resolution mass spectrometry.

Acute myocardial infarction (AMI) poses a grave threat to human life. However, most clinical biomarkers have limitations of low sensitivity and specificity. Therefore, screening novel glycan biomarkers with high sensitivity and specificity is crucial for the prevention and treatment of AMI. The novel method of ultrahigh-performance liquid chromatography coupled to quadrupole-Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) with d0/d5-BOTC probe labeling for relative quantification of glycans based on Pronase E digestion was established to screen novel glycan biomarkers in the serum of 34 AMI patients relative to healthy volunteers. The monosaccharide model D-glucosamine was used to investigate the effectiveness of the derivatization; the limit of detection (S/N = 3) was 10 amol. The accuracy was verified based on the consistency of different theoretical molar ratios (d0/d5 = 1:2, 2:1) and intensity ratios following digestion of glycoprotein ribonuclease B. Expressions of H4N4F3SA, H4N6F2, H4N6SA, H4N6F3 and H5N4FSA in the serum were significantly different (p < 0.0005) between AMI patients and healthy volunteers. The area under the receiver operating characteristic curve (AUC) for H4N6SA, H5N4FSA, and H4N6F2 was greater than 0.9039. Based on the proposed method, H4N6SA, H5N4FSA, and H4N6F2 in human serum showed high accuracy and specificity and may serve as potential glycan biomarkers, crucial for the diagnosis and treatment monitoring of AMI.

A Unique Case of Membranous Lupus Nephritis Identified After Pronase Digestion.

Membranous nephropathy (MN) is a common etiology of nephrotic syndrome (NS) in Caucasian adults. With treatment strategies heavily dependent on differentiating between primary versus secondary MN, tissue diagnosis remains paramount in the setting of indeterminant serological studies and remains the gold standard. Direct immunofluorescence on frozen sections remains standard practice, though with inadequate kidney tissue, antigen retrieval with proteases on formalin-fixed paraffin-embedded tissue can be a viable alternative for direct immunofluorescence. We report a patient who presented with nephrotic syndrome, indeterminant serological workup including primary antigen phospholipase-2 receptor antibody (PLA2R). Histology revealed a membranous pattern of injury with a negative standard panel of immunocomplex deposits on direct immunofluorescence. Upon re-examination of paraffin-embedded tissue via protease processing, Immunofluorescence unmasked membranous lupus nephritis. This case highlights the possibility of negative direct immunofluorescence on viable frozen tissue which is unmasked after protease treatment on formalin-fixed paraffin-embedded tissue sample revealing immunocomplex deposits.

The effect of chemical treatment of the sheep embryo zona pellucida on the ability of blastocysts to hatch after vitrification and warming.

BACKGROUND: The embryo release from the zona pellucida is of prerequisites of successful implantation. OBJECTIVES: Regarding the negative impact of embryo cryopreservation on the blastocysts hatchability, the aim of the present study was to investigate the effects of treating embryonic zona pellucida with pronase or acidic Tyrode's solution (ATS) before morula formation on the viability, freezability, and hatchability of vitrified-warmed resulted blastocysts. METHODS: In the first experiment, the zona pellucida of 3- and 4-day-old embryos were treated with the above compounds for 30 or 45 s. Then, the competency of the treated embryos to reach to blastocyst stage and the hatchability of resulting blastocysts were investigated. In the second experiment, the cryo-survivability and hatching rate of blastocysts resulting from 3-day-old embryos treated with pronase and ATS for 30 s were tested. RESULTS: In the first experiment and in contrast to the 45 s exposure, 30-s exposure of embryos to pronase or ATS did not have negative effect on the viability and development of embryos to blastocyst stage. In the second experiment, the freezability of blastocysts derived from 3-day-old embryos treated with pronase and ATS for 30 s was not different from that of the control group. However, the hatching rate of the pronase group was significantly higher than that of the control group. CONCLUSION: The results of the present study showed that reducing the thickness of zona pellucida of sheep embryos with pronase had no negative effect on the developmental competency and freezability of the treated embryos and improved the hatchability of vitrified-warmed blastocysts.

Relative quantitation of glycans in cetuximab using ultra-high-performance liquid chromatography-high-resolution mass spectrometry by Pronase E digestion.

Glycans play important roles in the activity and function of monoclonal antibodies (mAbs). In this study, an isotope labeling method for the relative quantitative analysis of glycans in cetuximab, a chimeric human/mouse IgG1 monoclonal antibody that specifically targets epidermal growth factor receptor, via hydrophilic interaction LC-ultra-high-performance LC-HRMS was established based on Pronase E digestion. To this aim, novel isotope MS probes, i.e., 3-benzoyl-2-oxothiazolidine-4-carboxylic acid (d0-BOTC) and 3-(2,3,4,5,6-pentadeuterio-benzoyl)-2-oxothiazolidine-4-carboxylate acid (d5-BOTC), which include a carboxyl group to target the amino functional group in glycosylamine, were developed. The nonspecific Pronase E enzyme could simultaneously digest the peptide bound to the N- and O-glycans into glycosylamine having only one amino acid. Since the mass difference between the light- and heavy-labeled glycans was 5.0 Da, the relative abundance of their MS peaks was used to achieve the qualitative and relative quantitative analysis of glycans. Sialylglycopeptide was used as a complex glycan model to validate the accuracy of the method. The results demonstrated the good linearity (R2 ≥ 0.9994) between the experimentally detected MS intensity ratios and the theoretical molar ratios of the d0-BOTC to the corresponding d5-BOTC derivatives in the dynamic range of 0.03-10 and 0.03-20 of three orders magnitude for the d5-BOTC/d0-BOTC ratios. The reproducibility was between 0.16% and 10.70%, and the limit of detection was 13 fmol. The feasibility of the relative quantification method was investigated by analyzing the glycan content in cetuximab, finding good consistency between experimental and theoretical molar ratios (5:1, 3:1, 1:1, 1:3, 1:5) of d0/d5-BOTC-labeled glycans. Finally, 13 glycans were successfully identified in cetuximab by applying this method using an in-house Tracefinder database. This study provides a novel strategy for the high throughput analysis, identification, and functional study of glycans in mAbs.

Nasopharyngeal cryptococcosis in a cat: interlaboratory variation in cryptococcal antigen assay test results.

Case summary: An indoor-only 6-year-old spayed female domestic cat was evaluated for a history of stertorous respiration. Skull radiographs revealed increased soft tissue density within the caudal aspect of the left nasal cavity. CT and rhinoscopy revealed a mass lesion in the choana, plus a smaller lesion, nearly completely occluding flow through the nasal passages. Rhinoscopy was used to collect a biopsy specimen from a fleshy, tan-yellow mass visualized in the caudal nasopharynx. Histopathology was diagnostic for Cryptococcus species infection and systemic antifungal therapy with fluconazole was initiated. Following a series of discordant results, serum samples were submitted to a veterinary diagnostic laboratory that utilized a cryptococcal antigen latex agglutination system with pretreatment of serum with pronase. Twenty-three months after the initial diagnosis, the cat's serum cryptococcal antigen titer declined to 1:5 and the cat has responded well to continuing treatment. Relevance and novel information: This case illustrates challenges associated with discordant test results for cryptococcal antigen among laboratories. Discordancies may be due to differences in assay design, or the underlying disease state itself, or whether serum is pre-treated with pronase; with some tests relying on the training and experience of the operator if the cryptococcal antigen detection test requires a subjective interpretation. It also resolves some confusion in the literature related to the assay types available and terminology used to describe them, and emphasizes the importance of considering cryptococcosis as an important differential for cats with upper respiratory signs, without nasal discharge, even if the cat is kept exclusively indoors.

New Mutants of Epsilon Toxin from Clostridium perfringens with an Altered Receptor-Binding Site and Cell-Type Specificity.

Epsilon toxin (Etx) from Clostridium perfringens is the third most potent toxin after the botulinum and tetanus toxins. Etx is the main agent of enterotoxemia in ruminants and is produced by Clostridium perfringens toxinotypes B and D, causing great economic losses. Etx selectively binds to target cells, oligomerizes and inserts into the plasma membrane, and forms pores. A series of mutants have been previously generated to understand the cellular and molecular mechanisms of the toxin and to obtain valid molecular tools for effective vaccination protocols. Here, two new non-toxic Etx mutants were generated by selective deletions in the binding (Etx-ΔS188-F196) or insertion (Etx-ΔV108-F135) domains of the toxin. As expected, our results showed that Etx-ΔS188-F196 did not exhibit the usual Etx binding pattern but surprisingly recognized specifically an O-glycoprotein present in the proximal tubules of the kidneys in a wide range of animals, including ruminants. Although diminished, Etx-ΔV108-F135 maintained the capacity for binding and even oligomerization, indicating that the mutation particularly affected the pore-forming ability of the toxin.

Effectiveness of chitosan against mature biofilms formed by food related bacteria.

Chitosan has proven antimicrobial properties against planktonic cell growth. Little is known, however, about its effects on already established biofilms. Oriented for application in food industry disinfection, the effectiveness of both medium molecular weight (MMW) chitosan and its enzymatically hydrolyzed product was tested against mature biofilms of four pathogenic strains, Listeria monocytogenes, Bacillus cereus, Staphylococcus aureus and Salmonella enterica, and a food spoilage species, Pseudomonas fluorescens. Unexpectedly, log reductions were in some cases higher for biofilm than for planktonic cells. One hour exposure to MMW chitosan (1% w/v) caused a 6 log viable cell reduction on L. monocytogenes monospecies mature biofilms and reduced significantly (3-5 log reductions) the attached population of the other organisms tested, except S. aureus. Pronase-treated chitosan was more effective than MMW chitosan on all tested microorganisms, also with the exception of S. aureus, offering best results (8 log units) against the attached cells of B. cereus. These treatments open a new possibility to fight against mature biofilms in the food industry.

Impact of rituximab on the T-cell flow cytometric crossmatch.

Rituximab is frequently used in the setting of ABO-incompatible renal transplants, and highly sensitized patients. Its interference with B-cell flow cytometric crossmatch (B-FCXM) is well known. However, its effect on the T-cell flow cytometric crossmatch (T-FCXM) has not been described. We aimed to evaluate the effect of rituximab on the T-FCXM using non-pronase and pronase treated donor lymphocytes and compare results with the single antigen bead (SAB) assay. In this retrospective study, 28 patients on rituximab therapy were evaluated against 30 donors. Using non-pronase treated donor lymphocytes, all 30 FCXMs showed strong B-cell positivity {median (IQR) B-cell ratio: 184.65 (253.17)} which significantly reduced {1.0 (1.18); p < 0.00001} with pronase treatment. 'T-cell tailing' phenomenon was observed in 17/30 FCXMs in the non-pronase group as a 'tail of T-cells', indicating a rare sub-population. However, it disappeared in the pronase-treated group. SAB assay did not show donor-specific antibodies (DSA) in all 17 patients with 'T-cell tailing' phenomenon. Although, rituximab is described to impact only B-FCXM, we have consistently found 'T-cell tailing' in 57% of T-FCXMs, which clears with pronase treatment. The 'T-cell tailing' led to weak positive T-FCMX ratios due to increased MFI in the FL1 channel. However, the absence of DSA in all recipients reinforces the fact that this is a false positive finding and should not be misconstrued as a possible class I DSA. Structural homology of Fc receptors on activated T-cells to CD20 could be a possible explanation of the same and provide insight into a novel mechanism of action of rituximab.

Impact of Pronase, Sodium Thiosulfate, and Methylene Blue Combinations on Development and Survival of Sodium Hypochlorite Surface-Disinfected Zebrafish (Danio rerio) Embryos.

Embryo surface disinfection is utilized in aquaculture to decrease the risk of pathogen introduction into established colonies. Zebrafish embryos are commonly disinfected with unbuffered sodium hypochlorite at 25-50 ppm for 10 min with or without concurrent treatment with chemicals, including pronase (Pron), sodium thiosulfate, and/or methylene blue; however, the impact of these chemicals on embryo survival and development has not been evaluated. In this study, AB and casper embryos were exposed to disinfection protocols that used Pron, sodium thiosulfate, and/or methylene blue (given alone, in various combinations, or all three combined) with 50 and 100 ppm sodium hypochlorite performed 6 and 24 h postfertilization (HPF). All groups were evaluated for survival, hatching, and malformations at 5 days postfertilization. Maximal survival (69%-97%) and hatching rates (66%-94%) were generally observed with sodium hypochlorite disinfection followed by exposure to both Pron and sodium thiosulfate and maintenance in standard embryo medium without methylene blue. Methylene blue had variable effects on survival and hatching. Higher survival and hatching rates were seen in AB embryos disinfected at 6 HPF and casper embryos disinfected at 24 HPF. Susceptibility to sodium hypochlorite toxicity differed by strain, emphasizing the need to test disinfection protocols on small embryo cohorts.

Efficiency of purification methods on the recovery of exopolysaccharides from fermentation media.

De-Man Rogosa and Sharpe (MRS) is a complex medium commonly used to obtain exopolysaccharides (EPS) from lactic acid bacteria. However, the various nutrients (carbon and nitrogen sources) of media and supplements added to enhance the bacterial growth and EPS production, may interfere with the purification of the extracts resulting in an over-estimation of the EPS and erroneous structural assignments. The efficiency of trichloroacetic acid (TCA)/pronase and 5-sulfosalicylic acid - SSA methods was evaluated. In addition, the interference of the major MRS broth components as well as lactose was evaluated using xanthan gum as model control EPS. It was concluded that MRS medium is a major source of interfering compounds in the quantification of EPS, mainly glucose-rich material and to a lesser extent mannoproteins from yeast extract. This effect was found to be potentiated by the presence of lactose. TCA/pronase method was shown to be more efficient in eliminating interferents.