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.

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.

Factor B Is the Second Lipopolysaccharide-binding Protease Zymogen in the Horseshoe Crab Coagulation Cascade.

Factor B is a serine-protease zymogen in the horseshoe crab coagulation cascade, and it is the primary substrate for activated factor C, the LPS-responsive initiator of the cascade. Factor C is autocatalytically activated to α-factor C on LPS and is artificially converted to ß-factor C, another activated form, by chymotrypsin. It is not known, however, whether LPS is required for the activation of factor B. Here we found that wild-type factor B expressed in HEK293S cells is activated by α-factor C, but not by ß-factor C, in an LPS-dependent manner and that ß-factor C loses the LPS binding activity of factor C through additional cleavage by chymotrypsin within the N-terminal LPS-binding region. Surface plasmon resonance and quartz crystal microbalance analyses revealed that wild-type factor B binds to LPS with high affinity comparable with that of factor C, demonstrating that factor B is the second LPS-binding zymogen in the cascade. An LPS-binding site of wild-type factor B was found in the N-terminal clip domain, and the activation rate of a clip domain deletion mutant was considerably slower than that of wild-type factor B. Moreover, in the presence of LPS, Triton X-100 inhibited the activation of wild-type factor B by α-factor C. We conclude that the clip domain of factor B has an important role in localizing factor B to the surface of Gram-negative bacteria or LPS released from bacteria to initiate effective proteolytic activation by α-factor C.

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.

The N-terminal Arg residue is essential for autocatalytic activation of a lipopolysaccharide-responsive protease zymogen.

Factor C, a serine protease zymogen involved in innate immune responses in horseshoe crabs, is known to be autocatalytically activated on the surface of bacterial lipopolysaccharides, but the molecular mechanism of this activation remains unknown. In this study, we show that wild-type factor C expressed in HEK293S cells exhibits a lipopolysaccharide-induced activity equivalent to that of native factor C. Analysis of the N-terminal addition, deletion, or substitution mutants shows that the N-terminal Arg residue and the distance between the N terminus and the tripartite of lipopolysaccharide-binding site are essential factors for autocatalytic activation, and that the positive charge of the N terminus may interact with an acidic amino acid(s) of the molecule to convert the zymogen into an active form. Chemical cross-linking experiments indicate that the N terminus is required to form a complex of the factor C molecules in a sufficiently close vicinity to be chemically cross-linked on the surface of lipopolysaccharides. We propose a molecular mechanism of the autocatalytic activation of the protease zymogen on lipopolysaccharides functioning as a platform to induce specific protein-protein interaction between the factor C molecules.

Toll-like receptor 2 heterodimers, TLR2/6 and TLR2/1 induce prostaglandin E production by osteoblasts, osteoclast formation and inflammatory periodontitis.

TLR2 forms heterodimers with TLR1 and TLR6, and regulates host defense mechanisms against pathogens. We examined the role of TLR2 heterodimer signaling in osteoclast formation and inflammatory periodontitis. In co-cultures of mouse bone marrow cells and osteoblasts, a TLR2/6 ligand (diacylated lipopeptide designed from Gram-positive bacteria) markedly induced osteoclast formation. A TLR2/1 ligand (triacylated lipopeptide designed from Gram-negative bacteria) also induced osteoclast formation. The osteoclast formation induced by TLR2/6 and TLR2/1 ligands was completely suppressed by indomethacin. Osteoblasts expressed TLR1, 2, 4, and 6 mRNAs, and both TLR2/6 and TLR2/1 ligands induced the expression of COX-2, mPGES-1, and RANKL mRNA, as well as PGE production in osteoblasts. Both TLR2/6 and TLR2/1 ligands induced the resorption of mandibular alveolar bone in organ cultures, and elicited inflammatory periodontitis in vivo. Therefore, TLR2 heterodimer signaling may play a key role in PGE-mediated inflammatory bone loss in periodontal disease.

Soluble cell wall beta-glucan of Candida induces/enhances apoptosis and oxidative stress in murine lung.

The bioactivity of cell wall component(s) of fungi has not been fully investigated, especially in vivo. We have successfully extracted a soluble cell wall beta-glucan from C. albicans (CSBG), and shown that pulmonary exposure to CSBG induces lung inflammation in mice. CSBG-induced lung inflammation was concomitant with the nuclear translocation of signal transducer and activator of transcription (STAT)6 and enhanced lung expression of various cytokines and chemokines. However, the effects of CSBG on the murine respiratory system and their mechanisms have not been fully investigated. In this study, we further explored the effects of pulmonary exposure to CSBG on lung pathophysiology in terms of the induction of apoptosis and enhancement of oxidative stress. ICR mice were intratracheally instilled with vehicle, CSBG, or structurally degraded products of CSBG by formic acid (DEG-CSBG), and 24 h later, the lungs were isolated and evaluated for apoptosis by the TUNEL assay and oxidative stress by immunohistochemistry of 8-hydroxy-2'-deoxyguanosine (8-OHdG), a proper marker of the oxidative DNA damage. In another experiment, the mice were sacrificed and lungs were isolated 4 h after the instillation with vehicle or CSBG for evaluation of transcriptional level of heme oxygenase (HO)-1, a stress responsive protein. Pulmonary exposure to CSBG significantly induced apoptosis and enhanced the formation of 8-OHdG in the lung parenchyma as compared to vehicle or DEG-CSBG exposure. CSBG significantly induced HO-1 in the lung. Taken together, CSBG induces/enhances apoptosis and oxidative stress in the lung in association with lung inflammation/injury.

Candida soluble cell wall beta-glucan facilitates ovalbumin-induced allergic airway inflammation in mice: Possible role of antigen-presenting cells.

BACKGROUND: Although fungi have been implicated as initiating/deteriorating factors for allergic asthma, their contributing components have not been fully elucidated. We previously isolated soluble beta-glucan from Candida albicans (CSBG) (Ohno et al., 2007). In the present study, the effects of CSBG exposure on airway immunopathology in the presence or absence of other immunogenic allergen was investigated in vivo, and their cellular mechanisms were analyzed both in vivo and in vitro. METHODS: In vivo, ICR mice were divided into 4 experimental groups: vehicle, CSBG (25 microg/animal), ovalbumin (OVA: 2 microg/animal), and CSBG + OVA were repeatedly administered intratracheally. The bronchoalveolar lavage cellular profile, lung histology, levels of cytokines and chemokines in the lung homogenates, the expression pattern of antigen-presenting cell (APC)-related molecules in the lung digests, and serum immunoglobulin values were studied. In vitro, the impacts of CSBG (0-12.5 microg/ml) on the phenotype and function of immune cells such as splenocytes and bone marrow-derived dendritic cells (BMDCs) were evaluated in terms of cell proliferation, the surface expression of APC-related molecules, and OVA-mediated T-cell proliferating activity. RESULTS: In vivo, repeated pulmonary exposure to CSBG induced neutrophilic airway inflammation in the absence of OVA, and markedly exacerbated OVA-related eosinophilic airway inflammation with mucus metaplasia in mice, which was concomitant with the amplified lung expression of Th2 cytokines and IL-17A and chemokines related to allergic response. Exposure to CSBG plus OVA increased the number of cells bearing MHC class II with or without CD80 in the lung compared to that of others. In vitro, CSBG significantly augmented splenocyte proliferation in the presence or absence of OVA. Further, CSBG increased the expression of APC-related molecules such as CD80, CD86, and DEC205 on BMDCs and amplified OVA-mediated T-cell proliferation through BMDCs. CONCLUSION: CSBG potentiates allergic airway inflammation with maladaptive Th immunity, and this potentiation was associated with the enhanced activation of APCs including DC.

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.

Pulmonary exposure to diesel exhaust particles enhances coagulatory disturbance with endothelial damage and systemic inflammation related to lung inflammation.

Pulmonary exposure to diesel exhaust particles (DEP) enhances lung inflammation related to bacterial endotoxin (lipopolysaccharide [LPS]) in mice. Severe lung inflammation can reportedly induce coagulatory abnormalities and systemic inflammation. This study examined the effects of components of DEP on lung inflammation, pulmonary permeability, coagulatory changes, systemic inflammatory response, and lung-to-systemic translocation of LPS in a murine model of lung inflammation. ICR mice were divided into six experimental groups that intratracheally received vehicle, LPS (2.5 mg/kg), organic chemicals in DEP (DEP-OC; 4 mg/kg) extracted with dicloromethane), residual carbonaceous nuclei of DEP (washed DEP: 4 mg/kg), DEP-OC + LPS, or washed DEP + LPS. Both DEP components exacerbated lung inflammation, vascular permeability, and the increased fibrinogen and E-selectin levels induced by LPS. With overall trends, the exacerbation was more prominent with washed DEP than with DEP-OC. Washed DEP + LPS significantly decreased activated protein C and antithrombin-III and elevated circulatory levels of interleukin (IL)-6, keratinocyte chemoattractant (KC), and LPS as compared with LPS alone, whereas DEP-OC + LPS elevated IL-6, KC, and LPS without significance. These results show that DEP components, especially washed DEP, amplify the effects if LPS on the respiratory system and suggest that they contribute to the adverse health effects of particulate air pollution on the sensitive populations with predisposing vascular and/or pulmonary diseases, including ischemic vascular diseases and respiratory infection.

[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.

Pulmonary toxicity induced by intratracheal instillation of Asian yellow dust (Kosa) in mice.

Asian yellow dust (Kosa) causes adverse respiratory health effects in humans. The objective of this study was to clarify the lung toxicity of Kosa. ICR mice (5 weeks of age) were administered intratracheally with Kosa samples-two samples from Maowusu desert and Shapotou desert, one sample consisted of Shapotou Kosa plus sulfate, and natural Asian dust (NAD) from the atmosphere of Beijing-at doses of 0.05, 0.10 or 0.20mg/mouse at four weekly intervals. The four Kosa samples tested had similar compositions of minerals and concentrations of elements. Instillation of dust particles caused bronchitis and alveolitis in treated mice. The magnitude of inflammation was much greater in NAD-treated mice than in the other particles tested. Increased neutrophils, lymphocytes or eosinophils in bronchoalveolar lavage fluids (BALF) of treated mice were dose dependent. The number of neutrophils in BALF at the 0.2mg level was parallel to the content of ß-glucan in each particle. The numbers of lymphocytes and eosinophils in BALF at the 0.2mg level were parallel to the concentration of SO(4)(2-) in each particle. Pro-inflammatory mediators-such as interleukin (IL)-12, tumor necrosis factor-(TNF)-α, keratinocyte chemoattractant (KC), monocyte chemotactic protein (MCP)-l and macrophage inflammatory protein-(MIP)-lα in BALF-were greater in the treated mice. Specifically, NAD considerably increased pro-inflammatory mediators at a 0.2mg dose. The increased amounts of MlP-lα and TNF-α at 0.2mg dose corresponded to the amount of ß-glucan in each particle. The amounts of MCP-l or IL-12 corresponded to the concentration of sulfate (SO(4)(2-)) at a 0.2mg dose. These results suggest that inflammatory lung injury was mediated by ß-glucan or SO(4)(2-), which was adsorbed into the particles, via the expression of these pro-inflammatory mediators. The results also suggest that the variations in the magnitude of inflammation of the tested Kosa samples depend on the amounts of these toxic materials.