Application of a Recombinant Three-Factor Chromogenic Reagent, PyroSmart, for Bacterial Endotoxins Test Filed in the Pharmacopeias.

Assays using lysate reagents prepared from horseshoe crab hemocyte extract (limulus amoebocyte lysate, LAL) are commonly and widely used to detect and measure endotoxin in parenteral drugs and medical devices. However, lysate reagents suffer from lot-to-lot variations leading to possible fluctuations in testing. Also, this continued usage of lysate reagents leads to the possible decline of the horseshoe crab population. Recently, a new recombinant chromogenic reagent, PyroSmart, consisting of three recombinant factors was introduced to the market. There are now three recombinant products; two with recombinant factor C reagents and PyroSmart with the complete recombinant LAL system. We evaluated the applicability of the reagent to the harmonized bacterial endotoxins test in the United States, European and Japanese pharmacopeias. The recombinant product showed equivalent potency of thirteen endotoxins from different bacterial strains to conventional chromogenic lysate reagents as long as their assay modes are identical. All analytical characteristics or assay parameters of the reagent satisfied the acceptance criteria which are set for the use for the bacterial endotoxins test filed in the pharmacopeias. All of 109 parenteral drugs tested can be measured with PyroSmart within respective maximum allowable dilutions. The lot-to-lot variation in recovery of endotoxin added in the parenteral drugs for PyroSmart was equal to or less than those of six limulus lysate reagents. In conclusion, the present study suggests that the recombinant reagent, PyroSmart, provide a good alternative to the LAL reagents with better lot-to-lot variation.

Changes of Absorptive and Secretory Transporting System of (1 → 3) ß-D-glucan Based on Efflux Transporter in Indomethacin-induced Rat.

Infection and inflammation suppress the expression and activity of several drug transporters in liver. In the intestine, P-glycoprotein (P-gp/MDR1), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP) are important barriers to the absorption of many clinically important drugs. The expression and activity of these proteins were examined under inflammation. Drug transport was determined in jejunum and ileum segments isolated from 1.0 mg/kg, 5.0 mg/kg, and 7.5 mg/kg indomethacin-treated or control rats in diffusion chambers. Transport of laminaran, used as a model compound of (1-3) ß-D-glucan, was measured for 120 min in the presence or absence of inhibitors. Reverse transcription-polymerase chain reaction was used to measure mRNA levels. Compared with controls, levels of Mdr1a mRNA were significantly decreased in the jejunum and ileum of 7.5 mg/kg indomethacin-treated rats. Both reductions in the basolateral to apical efflux of laminaran and increases in the apical to basolateral influx of laminaran were observed, resulting in significant increases in the apical to basolateral absorption of laminaran in 7.5 mg/kg indomethacin-treated rats. The inhibitory effect of verapamil on laminaran transport was observed in control rats but not in indomethacin-treated rats. Fluorescein isothiocyanate dextran 40,000 permeability, membrane resistance, and claudin-4 mRNA level were not altered, indicating no change in the paracellular pathway. These results indicate that indomethacin-induced inflammation reduces the intestinal expression and activity of P-gp in rats, which elicits corresponding changes in the intestinal transport of laminaran. Hence, inflammatory diseases may impose variability in drug bioavailability through alterations in the intestinal expression and activity of drug transporters.

Retraction Note to: Changes of Absorptive and Secretory Transporting System of (1 → 3) ß-D-glucan Based on Efflux Transporter in Indomethacin-induced Rat.

The Editor-in-Chief has retracted this article [1] based on an investigation by the Ministry of Education, Culture, Sports, Science and Technology, Japan, which found that the article contained overlap with a previously published article by Kalitsky-Szirtes J, et al. [2].

Genetic engineering approach to develop next-generation reagents for endotoxin quantification.

The bacterial endotoxin test, which uses amebocyte lysate reagents of horseshoe crab origin, is a sensitive, reproducible and simple assay to measure endotoxin concentration. To develop sustainable raw materials for lysate reagents that do not require horseshoe crabs, three recombinant protease zymogens (factor C, derived from mammalian cells; factor B; and the proclotting enzyme derived from insect cells) were prepared using a genetic engineering technique. Recombinant cascade reagents (RCRs) were then prepared to reconstruct the reaction cascade in the amebocyte lysate reagent. The protease activity of the RCR containing recombinant factor C was much greater than that of recombinant factor C alone, indicating the efficiency of signal amplification in the cascade. Compared with the RCR containing the insect cell-derived factor C, those containing mammalian cell-derived factor C, which features different glycosylation patterns, were less susceptible to interference by the injectable drug components. The standard curve of the RCR containing mammalian cell-derived recombinant factor C had a steeper slope than the curves for those containing natural lysate reagents, suggesting a greater sensitivity to endotoxin. The present study supports the future production of recombinant reagents that do not require the use of natural resources.

[Evaluation of an improved pretreatment method for the measurement of (1-->3)-beta-D-glucan in blood samples].

Measurement of blood (1-->3)-beta-D-glucan is useful for early diagnosis and follow-up of the therapeutic process of deep seated mycoses. The Fungitec G test MK (Seikagaku Corp., Tokyo) kit using alkaline-pretreatment followed by chromogenic kinetic assay has been widely used in Japan because of its high sensitivity and easy handling of a large number of samples. Discrepancy in the levels of (1-->3)-beta-D-glucan and/or in the quantitative judgement, however, has been pointed out between this kit and other commercial kits. One of the reasons for this discrepancy has been reported to be non-specific reactions caused by substances other than beta-glucan. In this study, we have improved the alkaline pretreatment reagent by changing the concentration of KOH and salts, resulting in a marked reduction of the non-specific reaction. Recovery of standard beta-glucan added to plasma or serum after the improved pretreatment was 80 to 120%, and no amidolytic activity was detected either in plasma or in serum. By the improved pretreatment, the incidence of non-specific reactions, i.e., those that exceed the quantitation limit (3.9 pg/mL), were markedly decreased from 139 to 16 out of 200 plasma samples and from 106 to 22 out of 170 serum samples. The incidence of strong non-specific reactions, i.e., those that exceed the cut-off level (20 pg/mL), were also decreased from seven to one with plasma and seven to zero with serum samples. Correlation between corrected beta-glucan measurements by the current pretreatment and non-corrected ones by the improved pretreatment was quite good. The improved method is thus expected to decrease the frequency of non-specific false-positive reactions, with the high sensitivity of Fungitec G test MK.

Nonlinear intestinal absorption of (1-->3)-beta-D-glucan caused by absorptive and secretory transporting system.

The mechanism of the nonlinear concentration dependence of intestinal absorption of (1-->3)-beta-D-glucan was studied using in situ rat intestinal perfusion, as well as the in vitro Ussing-type chamber method mounted with rat intestinal tissue. The intestinal absorption rate constant of a (1-->3)-beta-D-glucan, laminaran, evaluated by the loop method increased significantly with increasing concentration of laminaran up to 0.5 muM in a nonlinear fashion and tended to decrease at higher concentrations. Mucosal-to-serosal directed permeation of the laminaran across rat ileal sheets evaluated by the in vitro Ussing-type chamber method also decreased in a dose-dependent fashion. Serosal-to-mucosal directed permeation decreased in a concentration-dependent manner. In addition, the serosal-to-mucosal flux was reduced in the presence of metabolic inhibitor, 2,4-di-nitrophenol. These results suggest that laminaran is secreted into the intestinal lumen predominantly by the efflux transporting system. We conclude that intestinal transport of (1-->3)-beta-D-glucan involves specialized transporter or something similar in both absorptive and secretory directions, and complex nonlinear intestinal absorption characteristics can be ascribed to the participation of multiple transport mechanism.

Blocking effect of anti-Dectin-1 antibodies on the anti-tumor activity of 1,3-beta-glucan and the binding of Dectin-1 to 1,3-beta-glucan.

Schizophyllan (SPG) is used to treat cervical cancer in combination with irradiation to enhance the immunological surveillance system. Dectin-1 is a cell surface receptor for 1,3-beta-glucan. In this study, we prepared two anti-Dectin-1 monoclonal antibodies, 4B2 and SC30 having a K(D) of 7.04 x 10(-8) M and 1.55 x 10(-7) M, respectively, and evaluated the role of Dectin-1 in SPG-induced anti-tumor activity in mice. Expression of Dectin-1 on peritoneal macrophages and binding of SPG to the cells were decreased by administration of 4B2 and SC30. SPG-mediated anti-tumor activity was inhibited by 4B2 and SC30. 4B2 and SC30 inhibited the binding of SPG to splenocytes from mice. The binding of SPG-biotin to Dectin-1-transfected HEK293 cells was inhibited by 4B2, but not SC30. 4B2 and SC30 differ in their influence on Dectin-1 between primary cells and transduced cells, and Dectin-1 effects 1,3-beta-glucan-mediated anti-tumor activity in mice by binding to SPG.

Characterization of beta-glucan recognition site on C-type lectin, dectin 1.

Dectin 1 is a mammalian cell surface receptor for (1-->3)-beta-d-glucans. Since (1-->3)-beta-d-glucans are commonly present on fungal cell walls, it has been suggested that dectin 1 is important for recognizing fungal invasion. In this study we tried to deduce the amino acid residues in dectin 1 responsible for beta-glucan recognition. HEK293 cells transfected with mouse dectin 1 cDNA could bind to a gel-forming (1-->3)-beta-d-glucan, schizophyllan (SPG). The binding of SPG to a dectin 1 transfectant was inhibited by pretreatment with other beta-glucans having a (1-->3)-beta-d-glucosyl linkage but not by pretreatment with alpha-glucans. Dectin 1 has a carbohydrate recognition domain (CRD) consisting of six cysteine residues that are highly conserved in C-type lectins. We prepared 32 point mutants with mutations in the CRD and analyzed their binding to SPG. Mutations at Trp(221) and His(223) resulted in decreased binding to beta-glucan. Monoclonal antibody 4B2, a dectin- 1 monoclonal antibody which had a blocking effect on the beta-glucan interaction, completely failed to bind the dectin-1 mutant W221A. A mutant with mutations in Trp(221) and His(223) did not have a collaborative effect on Toll-like receptor 2-mediated cellular activation in response to zymosan. These amino acid residues are distinct from residues in other sugar-recognizing peptide sequences of typical C-type lectins. These results suggest that the amino acid sequence W221-I222-H223 is critical for formation of a beta-glucan binding site in the CRD of dectin 1.