Characterization of AEBSF-antibody modifications for a protease inhibitor supplementation strategy.

Application of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), during the cell culture process was demonstrated to effectively reduce proteolytic activity at a specific amino acid site during the production of an HIV-1 broadly neutralizing antibody (bNAb). However, the addition of AEBSF could potentially introduce some modifications to the bNAb protein. Experimental design from sample preparation to LC-MS characterization was performed using middle-up and bottom-up approaches to identify AEBSF-modified species for the bNAb using an AEBSF supplementation in the cell culture media. Modified species along with the unmodified control sample were also subjected to binding activity assessment. The results showed that two amino acids (Tyr177 and Lys250) were susceptible to AEBSF modification in the bNAb test articles but at a negligible level and not in the CDR regions, which therefore did not reduce the in vitro binding activity of the bNAb.

A monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay for flubendiamide detection.

Flubendiamide (FD), the first commercial phthalic acid diamide that targets insect ryanodine receptor (RyRs), has played an important role in pest management. With its extensive worldwide application, a rapid and convenient method to detect its existence in the environment is necessary. In this study, an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to analyse FD residue on environmental and food samples. The established icELISA showed a half maximal inhibition concentration (IC50) of 17.25 µg L-1, with a working range of 4.06-103.59 µg L-1 for FD, and showed no cross-reactivity with chlorantraniliprole, cyantraniliprole, and several FD analogues. Average FD recoveries from spinach, tap water, and soil samples were 89.3-112.3%, 93.0-102.1%, and 86.9-97.6%, respectively. Meanwhile, FD detection results of icELISA were compared with those of ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The comparable results verified that icELISA was suitable for rapid detection of FD residue in environmental and agricultural samples.

Incidence of Hypersensitivity Reactions During Hemodialysis.

BACKGROUND/AIMS: A recent alert from Spanish health authorities warned of a higher incidence of reported hypersensitivity reactions to hemodialysis membranes with polysulfone, in the 2017 review of acute reactions to dialyzers found only published reports in the 21st century on polysulfone and its derivatives. The aim is to assess/evaluate the current incidence and characteristics of hypersensitivity reactions in hemodialysis patients. METHODS: A retrospective multicentre study in 9 Spanish hospitals evaluated patients in whom a hypersensitivity reaction required a change in dialyzer membrane. RESULTS: A total of 37 patients out of 1561 (2.37%) had hypersensitivity reactions and clinical, epidemiological and analytical data were available for 33 patients (2.11%). The membranes involved were polysulfone (n=23), polynephron (n=8), polyethersulfone (n=1) and polyacrylonitrile (n=1). This distribution reflected the frequency of use of membranes in the participating dialysis units. The reactions were described as type A in 18 cases and type B in 15 cases. There were no significant differences between the two types in clinical symptoms, the composition of the membrane involved, the method of sterilization, the season, or the time during the session in which they occurred. The most frequent symptom was dyspnea/breathlessness (64% of reactions). Eosinophilia was common (74%). 54% of the reactions occurred within the first 30 minutes of hemodialysis, 64% occurred during the first year of dialysis, and 54% required discontinuation of dialysis session. Cellulose triacetate was used as an alternative dialyzer in 78% of the cases. CONCLUSION: The incidence of hypersensitivity reactions was in the range found in reports from 20 years ago and is observed associated with synthetic membranes, not just polysulfones. Cellulose triacetate appears to be a good alternative for these patients.

Mechanisms Involved in Hypersensitivity Reactions to Polysulfone Hemodialysis Membranes.

Several cases of patients with anaphylactic or systemic hypersensitivity reactions to polysulfone (PS) hemodialysis (HD) membranes and tolerance to cellulose triacetate (CTA) membranes have recently been reported. To investigate the mechanisms involved in PS hypersensitivity, basophil, T cell, and complement activation were analyzed in acute-phase samples from two patients with systemic reactions to PS-based membranes. Basophil and T cell activation, as well as higher serum tryptase levels were detected in acute-phase samples compared with basal levels. Complement levels (C3 and C4) were decreased in acute-phase samples from PS-allergic patients to a higher extent than in samples from control donors taken at the same time points, indicating complement activation during the acute reactions. An experimental external circuit was established on pediatric membranes after rinsing with low or high priming volumes of saline solution, to analyze basophils, T cells, and complement activation in blood samples from 10 PS-allergic and 8 nonallergic HD patients upon contact with PS-based or CTA membranes. Predialysis and postdialysis samples were collected. Basophils from PS-allergic patients exhibited increased degranulation, and T cells showed significantly increased activation after contact with PS-based membranes primed with low volumes of saline. No activation was detected in leukocytes from nonallergic patients under the same experimental conditions. Membrane priming with high volumes of saline abrogated activation of basophils and T cells. However, basophils from allergic donors showed significantly higher responses to Fcεc stimulation after contact with PS membranes. Basophil degranulation and elevated serum tryptase levels in allergic patients during acute reactions support the systemic activation of mast cells and basophils during hypersensitivity reactions to PS-based membranes. A leachable component of the membranes might be responsible for cell activation in some patients.

Self-adjuvanting C18 lipid vinil sulfone-PP2A vaccine: study of the induced immunomodulation against Trichuris muris infection.

Despite the importance of the adjuvant in the immunization process, very few adjuvants merge with the antigens in vaccines. A synthetic self-adjuvant oleic-vinyl sulfone (OVS) linked to the catalytic region of recombinant serine/threonine phosphatase 2A from the nematode Angiostrongylus costaricensis (rPP2A) was used for intranasal immunization in mice previously infected with Trichuris muris The animal intranasal immunization with rPP2A-OVS showed a reduction of 99.01% in the number of the nematode eggs and 97.90% in adult. The immunohistochemical analysis of the intestinal sections showed that in immunized animals with lipopeptide the mucus was significantly higher than in the other experimental groups. Also, these animals presented significantly different chemokine, CCL20 and CCL11, levels. However, although the number and size of Tuft cells did not vary between groups, the intensity of fluorescence per cell was significant in the group immunized with the rPP2A-OVS. The results of the present study suggest that mice immunized with the lipopeptide are capable of activating a combined Th17/Th9 response. This strategy of immunization may be of great applicability not only in immunotherapy and immunoprophylaxis to control diseases caused by nematodes but also in pathologies necessitating action at the level of the Th9 response in the intestinal mucosa.

Lifitegrast: First LFA-1/ICAM-1 antagonist for treatment of dry eye disease.

Dry eye disease is an extremely common condition affecting millions worldwide. The underlying pathophysiological mechanism is thought to be localized inflammation of the ocular surface resulting in the localization of T cells at this surface followed by their activation and subsequent liberation of cytokines. This effect on T cells results from the binding of lymphocyte function-associated antigen-1 (LFA-1) located on T cells to intercellular adhesion molecule 1 (ICAM-1) expressed on inflamed epithelium and endothelium, and on T cells. Lifitegrast is a T-cell integrin antagonist designed to mimic ICAM-1, thus blocking the interaction of LFA-1 and ICAM-1. Lifitegrast enters the systemic circulation to a limited extent thus reducing the likelihood of unwanted systemic reactions. Clinical trials in over 2,500 subjects with dry eye disease have shown that 5.0% lifitegrast given by ocular instillation causes a significant reduction in objective and subjective signs and symptoms of the disease. These beneficial effects are associated with a relatively low incidence of unwanted effects, almost all local in nature. In light of these findings, lifitegrast was approved by the Food and Drug Administration (FDA) in 2016 for the treatment of dry eye disease, the first drug with this mechanism of action to be so approved.

PI3K and ERK1/2 kinase inhibition potentiate protease inhibitor to attenuate allergen induced Th2 immune response in mouse.

Proteases affect immune response by activating PI3K, ERK1/2 and p38 kinase. In present study, therapeutic effect of PI3K, ERK1/2 and p38 kinase inhibitor in combination with serine protease inhibitor was evaluated in cockroach extract (CE) induced airway inflammatory disease. Mice were sensitized on day 0, 7 and 14 and challenged on day 27, 28 and 29 with CE. Mice were given PI3K, ERK1/2 and the p38 kinase inhibitor (iPI3K, iERK1/2 and the ip38) alone or with serine protease inhibitor 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF), 1h before challenge. On day 30 airway resistance of mice were determined and euthanized to collect blood, BAL fluid and lung for analysis. CE immunized mice showed PI3K, ERK1/2 and p38 kinase activation, increased airway resistance, cellular infiltration, Th2 cytokines IgE and IgG1. AEBSF given to mice reduced the CE induced allergic response. AEBSF given in combination of iPI3K/iERK1/2 reduced cellular infiltration in lungs. Furthermore, iPI3K/iERK1/2 with AEBSF significantly reduced the CE induced Th2 cytokines in comparison to monotherapy of kinase inhibitor and AEBSF (P<0.05). The combination of iPI3K/iERK1/2 with AEBSF enhanced IL-12 level that could further provide a mean of Th2 reduction. Best effect in reduction of allergic response in mice was observed on administration of AEBSF with iPI3K. Conclusively, the combination of PI3K kinase inhibitor with AEBSF reduced allergen induced airway response and has therapeutic potential for add-on therapy in allergic airway disease.

Methylsulfonylmethane inhibits NLRP3 inflammasome activation.

Methylsulfonylmethane (MSM) is an organosulfur compound and the health benefits associated with MSM include inflammation. Although MSM has been shown to have various physiological effects, no study has yet focused on inflammasome activation. The inflammasome is a multiprotein complex that serves as a platform for caspase 1-dependent proteolytic maturation and secretion of interleukin-1ß (IL-1ß). In this study, we tested the effect of MSM on inflammasome activation using mouse and human macrophages. In our results, MSM significantly attenuated NLRP3 inflammasome activation in lipopolysaccharide-primed macrophages, although it had no effect on NLCR4 or AIM2 inflammasome activation. Extracts of MSM-enriched vegetables presented the same inhibitory effect on NLRP3 inflammasome activation as MSM. MSM also attenuated the transcriptional expression of IL-1α, IL-1ß, IL-6, and NLRP3. Taken together, these results show that MSM has anti-inflammatory characteristics, interrupts NLRP3 inflammasome activation, and inhibits pro-cytokine expression. We further confirmed the intracellular mechanism of MSM in relation to NLRP3 inflammasome activation, followed by comparison with that of DMSO. Both chemicals showed a synergic effect on anti-NLRP3 activation and attenuated production of mitochondrial reactive oxygen species (ROS). Thus, MSM is a selective inhibitor of NLRP3 inflammasome activation and can be developed as a supplement to control several metabolic disorders.

Acometimento hepático na síndrome sulfona dapsona-induzida durante tratamento de hanseníase / Hepatic involvement in sulfone dapsone-induced syndrome during treatment of leprosy

A hanseníase é uma doença endêmica no Brasil e constitui grave problema de saúde pública. Em números absolutos, o Brasil é o segundo país que mais registra novos casos da doença por ano no mundo. O tratamento da hanseníase compreende: quimioterapia específica, supressão dos surtos reacionais, prevenção de incapacidades físicas, reabilitação física e psicossocial. A síndrome sulfona é uma condição multissistêmica potencialmente grave que pode ocorrer durante o tratamento de algumas dermatoses, entre elas a hanseníase. Relatamos um caso de síndrome de hipersensibilidade à dapsona (SHD) em um paciente masculino, de 32 anos, ocorrida durante o tratamento de hanseníase multibacilar...

The biocompatibility and separation performance of antioxidative polysulfone/vitamin E TPGS composite hollow fiber membranes.

The extended interaction of blood with certain materials like hemodialysis membranes results in the activation of cellular element as well as inflammatory response. This results in hypersensitive reactions and increased reactive oxygen species, which occurs during or immediately after dialysis. Although polysulfone (Psf) hollow fiber has been commercially used for acute and chronic hemodialysis, its biocompatibility remains a major concern. To overcome this, we have successfully made composite Psf hollow fiber membrane consisting of hydrophilic/hydrophobic micro-domains of Psf and Vitamin E TPGS (TPGS). These were prepared by dry-wet spinning using 5, 10, 15, 20 wt% TPGS as an additive in dope solution. TPGS was successfully entrapped in Psf hollow fiber, as confirmed by ATR-FTIR and TGA. The selective skin was formed at inner side of hollow fibers, as confirmed by SEM study. In vitro biocompatibility and performance of the Psf/TPGS composite membranes were examined, with cytotoxicity, ROS generation, hemolysis, platelet adhesion, contact and complement activation, protein adsorption, ultrafiltration coefficient, solute rejection and urea clearance. We show that antioxidative composite Psf exhibits enhanced biocompatibility, and the membranes show high flux and high urea clearance, about two orders of magnitude better than commercial hemodialysis membranes on a unit area basis.

Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody.

Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed. Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL(-1), the calibration curve was linear from 5.0 to 40 ng mL(-1) (R(2)=0.952) with an IC(50) value of 18.2 ng mL(-1). In the extracts of 20 Chinese traditional drugs, the detection capability (CCbeta) of vardenafil was 0.08 mg g(-1), the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue. The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells.

The purpose of this study was to identify the effect of sildenafil citrate on IL-1beta-induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1? stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1beta-induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1beta-induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1?-induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.

Effect of sildenafil citrate on interleukin-1beta-induced nitric oxide synthesis and iNOS expression in SW982 cells

The purpose of this study was to identify the effect of sildenafil citrate on IL-1 beta induced nitric oxide (NO) synthesis and iNOS expression in human synovial sarcoma SW982 cells. IL-1 beta stimulated the cells to generate NO in both dose- and time-dependent manners. The IL-1 beta -induced NO synthesis was inhibited by guanylate cyclase (GC) inhibitor, LY83583. When the cells were treated with 8-bromo-cGMP, a hydrolyzable analog of cGMP, NO synthesis was increased upto 5-fold without IL-1 beta treatment suggesting that cGMP is an essential component for increasing the NO synthesis. Synoviocytes and chondrocytes contain strong cGMP phosphodiesterase (PDE) activity, which has biochemical features of PDE5. When SW982 cells were pretreated with sildenafil citrate (Viagra), a PDE5 specific inhibitor, sildenafil citrate significantly inhibited IL-1 beta -induced NO synthesis and iNOS expressions. From this result, we noticed that PDE5 activity is required for IL-1 beta -induced NO synthesis and iNOS expressions in human synovial sarcoma cells, and sildenafil citrate may be able to suppress an inflammatory reaction of synovium through inhibition of NO synthesis and iNOS expression by cytokines.

Etoricoxib tolerability in patients with hypersensitivity to nonsteroidal anti-inflammatory drugs.

BACKGROUND: Adverse reactions to nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly observed, particularly among patients with chronic urticaria or asthma. The identification of a safe and reliable alternative is a frequent problem in clinical practice. Our aim was to investigate the clinical tolerability of etoricoxib, a new selective cyclooxygenase-2 inhibitor, in a group of patients with well-established NSAID hypersensitivity. METHODS: We assessed 31 adults (21 women and 10 men) who reported one or more adverse reactions to NSAIDs, manifested as cutaneous, respiratory or anaphylactic symptoms. Sixteen of them reported reactions to a single NSAID (single reactors) and 15 to more than one NSAID (multiple reactors); the most frequently involved drug was acetylsalicylic acid. First, each patient underwent allergologic tests (skin and/or oral challenge tests) with culprit NSAIDs and then tolerability tests with increasing doses of etoricoxib up to 120 mg. All challenges were performed under single-blind, placebo-controlled conditions. RESULTS: NSAID hypersensitivity was diagnosed in all 31 patients: 3 displayed positive results to pyrazolone skin tests and the other 28 to challenges with culprit NSAIDs. None reacted to either placebos or etoricoxib. CONCLUSIONS: Etoricoxib seems to be a safe alternative for patients with well-demonstrated NSAID hypersensitivity.

Short-term tolerability of etoricoxib in patients with cutaneous hypersensitivity reactions to nonsteroidal anti-inflammatory drugs.

BACKGROUND: Etoricoxib is a novel cyclooxygenase 2 selective inhibitor. Until now, there has not been information in the literature about its tolerability in patients with a history of hypersensitivity reactions to nonsteroidal anti-inflammatory drugs (NSAIDs). OBJECTIVE: To determine the short-term tolerability of etoricoxib in patients with a history of cutaneous adverse reactions to NSAIDs. METHODS: Single-blind challenge testing was performed on 2 different days using placebo (talc) and etoricoxib. On the first day, 2 placebo capsules were administered 1 hour apart; 7 days later, each patient received divided doses of the total therapeutic dose of 90 mg of etoricoxib: 22.5 mg initially and 67.5 mg 1 hour later if no reactive symptoms were noted. RESULTS: Of 141 patients who underwent challenge testing with etoricoxib, only 2 (1.4%) had positive test results; both developed wheals on the extremities. These 2 patients were treated with chlorpheniramine maleate (10 mg intravenously), and the symptoms completely resolved within 2 hours. None of the patients experienced adverse reactions to the placebo challenge. CONCLUSION: The low rate of adverse reactions to etoricoxib, tested by oral challenge, suggests that patients with previous cutaneous hypersensitivity reactions to NSAIDs (primarily urticaria and angioedema) may tolerate this drug.

Induction of interleukin-8 production via nuclear factor-kappaB activation in human intestinal epithelial cells infected with Vibrio vulnificus.

Vibrio vulnificus, a Gram-negative estuarine bacterium, is a causative agent of food-borne diseases, such as life-threatening septicaemia and wound infection disease. V. vulnificus penetrating into the epithelial barrier stimulates an inflammatory response in the adjacent mucosa. Therefore, interaction between V. vulnificus and epithelial cells is important for understanding of both the immunology of mucosal surfaces and V. vulnificus. In this study, we investigated the effect and action mechanism of V. vulnificus infection on production of interleukin (IL)-8, a proinflammatory cytokine, in human intestinal epithelial INT-407 cells. V. vulnificus infection significantly induced IL-8 production in a time- and multiplicity of infection (MOI)-dependent manner, as determined by human IL-8 enzyme-linked immunosorbent assay (ELISA). In addition, V. vulnificus infection significantly increased IL-8 mRNA levels in INT-407 cells, indicating that the increased IL-8 production by V. vulnificus occurred at the transcriptional level. V. vulnificus infection also enhanced IL-8 gene promoter activity in INT-407 cells transiently transfected with IL-8 promoter constructs, but this effect was impaired in INT-407 cells transfected with IL-8 promoter constructs deleted or mutated of a kappaB site. V. vulnificus infection increased the nuclear factor-kappaB (NF-kappaB) binding activity to a kappaB site and the degradation of IkappaB-alpha protein in a time- and a MOI-dependent manner. Furthermore, BAY11-7082, an inhibitor of NF-kappaB activation, significantly reduced the IL-8 production, NF-kappaB binding activity and IkappaB-alpha degradation induced by V. vulnificus infection. Taken together, these results indicate clearly that V. vulnificus infection significantly induces IL-8 production in human intestinal epithelial cells via NF-kappaB activation.

Role of p38 MAPK and NF-kB for chemokine release in coculture of human eosinophils and bronchial epithelial cells.

Eosinophils are principal effector cells of inflammation in allergic asthma, characterized by their accumulation and infiltration at inflammatory sites mediated by the chemokine eotaxin and their interaction with adhesion molecules expressed on bronchial epithelial cells. We investigated the modulation of nuclear factor-kappaB (NF-kappaB) and the mitogen-activated protein kinase (MAPK) pathway on the in vitro release of chemokines including regulated upon activation normal T cell expressed and secreted (RANTES), monokine induced by interferon-gamma (MIG), monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-8, and interferon-inducible protein-10 (IP-10) upon the interaction of human bronchial epithelial BEAS-2B cells and eosinophils. Gene expression of chemokines was evaluated by RT-PCR and the induction amount of chemokines quantified by cytometric bead array. NF-kappaB and p38 MAPK activities were assessed by electrophoretic mobility shift assay and Western blot, respectively. The interaction of eosinophils and BEAS-2B cells was found to up-regulate the gene expression of the chemokines IL-8, MCP-1, MIG, RANTES and IP-10 expression in BEAS-2B cells, and to significantly elevate the release of the aforementioned chemokines except RANTES in a coculture of BEAS-2B cells and eosinophils. IkappaB-alpha phosphorylation inhibitor, BAY 11-7082, and p38 MAPK inhibitor, SB 203580 could decrease the release of IL-8, IP-10 and MCP-1 in the coculture. Together, the above results show that the induction of the release of chemokines in a coculture of epithelial cells and eosinophils are regulated by p38 MAPK and NF-kappaB activities of BEAS-2B cells, at least partly, through intercellular contact. Our findings therefore shed light on the future development of more effective agents for allergic and inflammatory diseases.