Effect of Zr Content on Phase Stability, Deformation Behavior, and Young's Modulus in Ti-Nb-Zr Alloys.

Ti alloys have attracted continuing research attention as promising biomaterials due to their superior corrosion resistance and biocompatibility and excellent mechanical properties. Metastable ß-type Ti alloys also provide several unique properties such as low Young's modulus, shape memory effect, and superelasticity. Such unique properties are predominantly attributed to the phase stability and reversible martensitic transformation. In this study, the effects of the Nb and Zr contents on phase constitution, transformation temperature, deformation behavior, and Young's modulus were investigated. Ti-Nb and Ti-Nb-Zr alloys over a wide composition range, i.e., Ti-(18-40)Nb, Ti-(15-40)Nb-4Zr, Ti-(16-40)Nb-8Zr, Ti-(15-40)Nb-12Zr, Ti-(12-17)Nb-18Zr, were fabricated and their properties were characterized. The phase boundary between the ß phase and the α'' martensite phase was clarified. The lower limit content of Nb to suppress the martensitic transformation and to obtain a single ß phase at room temperature decreased with increasing Zr content. The Ti-25Nb, Ti-22Nb-4Zr, Ti-19Nb-8Zr, Ti-17Nb-12Zr and Ti-14Nb-18Zr alloys exhibit the lowest Young's modulus among Ti-Nb-Zr alloys with Zr content of 0, 4, 8, 12, and 18 at.%, respectively. Particularly, the Ti-14Nb-18Zr alloy exhibits a very low Young's modulus less than 40 GPa. Correlation among alloy composition, phase stability, and Young's modulus was discussed.

Corrosion behavior, in vitro and in vivo biocompatibility of a newly developed Ti-16Nb-3Mo-1Sn superelastic alloy.

The biocompatibility of a recently developed Ni-free Ti-16Nb-3Mo-1Sn (at.%) superelastic alloy was investigated both in vitro and in vivo. In addition, static water contact angle (WCA) and electrochemical tests were carried out. Commercial purity Ti (cp-Ti), which is already being used as a clinical material, was used as the control material. The alloy showed a stable corrosion behavior similar to that of the cp-Ti. The WCA measurements showed that the alloy exhibited hydrophilic properties that contributed to cell attachment to implants, as evident by the cytocompatibility tests. According to the in vivo implantation tests conducted on 30 adult BALB/c rats for periods up to 12 weeks, the tissue reaction around the implants was similar for both the cp-Ti and the alloy, and no significant difference was found in almost all parameters analyzed. Due to its stable superelastic properties accompanied with excellent biocompatibility and high corrosion resistance, we believe that this alloy is considered as a promising substitute for the biomedical materials containing Ni or other toxic elements.

Effect of Stoichiometry on Shape Memory Properties and Functional Stability of Ti⁻Ni⁻Pd Alloys.

Ti⁻Ni⁻Pd shape memory alloys are promising candidates for high-temperature actuators operating at above 373 K. One of the key issues in developing high-temperature shape memory alloys is the degradation of shape memory properties and dimensional stabilities because plastic deformation becomes more pronounced at higher working temperature ranges. In this study, the effect of the Ti:(Ni + Pd) atomic ratio in TixNi70-xPd30 alloys with Ti content in the range from 49 at.% to 52 at.% on the martensitic transformation temperatures, microstructures and shape memory properties during thermal cycling under constant stresses were investigated. The martensitic transformation temperatures decreased with increasing or decreasing Ti content from the stoichiometric composition. In both Ti-rich and Ti-lean alloys, the transformation temperatures decreased during thermal cycling and the degree of decrease in the transformation temperatures became more pronounced as the composition of the alloy departed from the stoichiometric composition. Ti2Pd and P phases were formed during thermal cycling in Ti-rich and Ti-lean alloys, respectively. Both Ti-rich and Ti-lean alloys exhibited superior dimensional stabilities and excellent shape memory properties with higher recovery ratio and larger work output during thermal cycling under constant stresses when compared with the alloys with near-stoichiometric composition.

Finding and analysing the minimum set of driver nodes required to control multilayer networks.

It is difficult to control multilayer networks in situations with real-world complexity. Here, we first define the multilayer control problem in terms of the minimum dominating set (MDS) controllability framework and mathematically demonstrate that simple formulas can be used to estimate the size of the minimum dominating set in multilayer (MDSM) complex networks. Second, we develop a new algorithm that efficiently identifies the MDSM in up to 6 layers, with several thousand nodes in each layer network. Interestingly, the findings reveal that the MDSM size for similar networks does not significantly differ from that required to control a single network. This result opens future directions for controlling, for example, multiple species by identifying a common set of enzymes or proteins for drug targeting. We apply our methods to 70 genome-wide metabolic networks across major plant lineages, unveiling some relationships between controllability in multilayer networks and metabolic functions at the genome scale.

Effect of annealing temperature on microstructure and superelastic properties of a Ti-18Zr-4.5Nb-3Sn-2Mo alloy.

In this study a new superelastic Ti-18Zr-4.5Nb-3Sn-2Mo alloy was prepared by adding 2at% of Mo as a substitute for Nb to the Ti-18Zr-11Nb-3Sn alloy, and heat treatment at different temperatures was conducted. The temperature dependence of superelasticity and annealing texture was investigated. Texture showed a dependence of annealing temperature: the specimen annealed at 923K for 0.3ks exhibited {113}ß<47¯1>ß type texture which was similar to the deformation texture, while specimens annealed at 973, 1073K, and 1173K showed {001}ß<110>ß type recrystallization texture which was preferable for recovery strain. The largest recovery strain of 6.2%, which is the same level as that of the Ti-18Zr-11Nb-3Sn alloy, was obtained in the specimen annealed at 1173K for 0.3ks due to the well-developed {001}ß<110>ß type recrystallization texture. The Ti-18Zr-3Nb-3Sn-2Mo alloy presented a higher tensile strength compared with the Ti-18Zr-11Nb-3Sn alloy when heat treated at 1173K for 0.3ks, which was due to the solid solution strengthening effect of Mo. Annealing at 923K for 0.3ks was effective in obtaining a good combination of a high strength as 865MPa and a large recovery strain as 5.6%. The high recovery strain was due to the high stress at which the maximum recovery stain was obtained which was attributed to the small grain size formed at low annealing temperature.

New possible biomarkers for diagnosis of infections and diagnostic distinction between bacterial and viral infections in children.

Detailed information about patients with infections is required to ensure appropriate choice of treatment. Although white blood cell (WBC) counts, and C-reactive protein (CRP) levels are useful diagnostic indicators of infections, more rapid and easily assayed indicator(s) could improve diagnosis. Moreover, it is of pivotal importance to distinguish bacteria or viruses as causative pathogens. Overall, TLR2 and TLR4 expression levels in neutrophils derived from individuals (n = 118) with bacterial (n = 37) and viral (n = 34) infections were higher than those in control samples (n = 47). Significant higher levels of TNF-α in patients with both types of the infection were observed, and those of IL-4, IL-8, IL-10, and IL-12 also were observed in the present study. Levels of IL-2, IL-8, and IL-10 on day 1 post-viral infection were significantly higher than those on day 1 post-bacterial infection. Therefore, there is a possibility that IL-4, IL-8, IL-10, IL-12 and TNF-α might be biomarkers for infections, in addition to WBC counts and CRP levels, and that IL-2, IL-8 or IL-10 are potentially able to distinguish between bacterial and viral infections.

An ATF4-Signal-Modulating Machine Other Than GADD34 Acts in ATF4-to-CHOP Signaling to Block CHOP Expression in ER-Stress-Related Autophagy.

Cells respond to ER-stress via ER-stress sensors, leading to the UPR and subsequent apoptosis; however, occasionally, they activate autophagy without subsequent apoptosis in response to ER-stress. We previously showed that the induction of apoptosis by ER-stress was related to the presence or absence of CHOP expression; nevertheless, how ATF4 expression is elicited without downstream CHOP expression is unknown. We studied the role of GADD34 on the induction of autophagy and/or apoptosis by NaF- or tunicamycin-induced ER-stress in HepG2 cells transfected with GADD34 siRNA. Although NaF and tunicamycin both induced PERK activation followed by eIF2α phosphorylation and ATF4 expression, CHOP expression was only induced by tunicamycin. Concomitant with the signaling change, autophagy was activated both by NaF and tunicamycin, and apoptosis was induced only by tunicamycin. After 4 h, GADD34 mRNA expression was also increased by NaF and tunicamycin. Suppression of GADD34 by GADD34 siRNA increased ATF4 expression in both NaF- and tunicamycin-treated cells. The GADD34 siRNA increased CHOP expression, which corresponded to increased ATF4 in tunicamycin-treated cells; however, the increased ATF4 did not induce CHOP expression in NaF-treated cells. In concert with signal changes, siRNA treatment additively increased the autophagic activity of both NaF- and tunicamycin-treated cells; however, apoptosis was produced and accelerated only for tunicamycin-treated cells. These findings indicate that GADD34 expression induced by ER-stress delays CHOP expression and retards apoptotic cell death, and that an ATF4-signal-modulating machine other than GADD34 acts on ATF4-to-CHOP signaling to block ATF4-induced CHOP expression in ER-stress related autophagy.

Novel Ti-base superelastic alloys with large recovery strain and excellent biocompatibility.

In this study, a new Ti-Zr-Nb-Sn alloy system was developed as Ni-free biomedical superelastic alloys with a large recovery strain and excellent biocompatibility. Ti-18Zr-(9-16)Nb-(0-4)Sn alloys were prepared by an Ar arc melting method and the effect of composition on the crystal structure and superelastic properties was investigated. A large superelastic recovery strain of 6.0% was observed in Ti-18Zr-12.5Nb-2Sn, Ti-18Zr-11Nb-3Sn, and Ti-18Zr-9.5Nb-4Sn alloys subjected to cold-rolling and solution treatment. XRD results showed that the large recovery strain of Sn-added alloys is due to a combination effect of a large transformation strain and a strong recrystallization texture. The Ti-18Zr-11Nb-3Sn alloy exhibited excellent cyclic stability with an extremely narrow stress hysteresis about 20MPa. Cytocompatibility was also examined using three types of cell lines, murine fibroblast L929, human osteosarcoma SaOS-2, and human umbilical vein endothelial cell HUVEC and the results showed that the Ti-18Zr-11Nb-3Sn alloy exhibited larger cell covering ratios when compared with those of the Ti-50.5Ni alloy for all kinds of cells.

Superelastic properties of biomedical (Ti-Zr)-Mo-Sn alloys.

A new class of Ti-50Zr base biomedical superelastic alloys was developed in this study. The (Ti-Zr)-Mo-Sn alloys exhibited a shape memory effect and superelastic property by adjusting Mo and Sn contents. The (Ti-Zr)-1.5Mo-3Sn alloy revealed the most stable superelasticity among (Ti-Zr)-(1-2)Mo-(2-4)Sn alloys. The superelastic recovery strain showed a strong dependence on heat treatment temperature after cold working in the (Ti-Zr)-1.5Mo-3Sn alloy. The superelastic recovery strain increased as the heat treatment temperature increased although the critical stress for slip decreased. The (Ti-Zr)-1.5Mo-3Sn alloy heat treated at 1073K exhibited excellent superelastic properties with a large recovery strain as large as 7% which is due to the strong {001}ß<110>ß recrystallization texture.

Gene expression profiling of lung myofibroblasts reveals the anti-fibrotic effects of cyclosporine.

Cyclosporine, a calcineurin inhibitor, is a potent immunosuppressive agent that acts chiefly through the inactivation of T-lymphocytes. Several clinical studies have demonstrated the effectiveness of cyclosporine for treating fibrotic lung disease, but the underlying mechanism remains elusive. We hypothesized that cyclosporine exerts direct effects against fibrogenesis of lung myofibroblasts, and aimed to elucidate the mechanism of this anti-fibrotic effect through gene-expression profiling using DNA microarray analysis. We found that cyclosporine suppressed the expression of alpha-smooth muscle actin and collagen type I in myofibroblasts that had been differentiated from a fetal human lung fibroblast cell line by induction with transforming growth factor (TGF)-ß. Furthermore, microarray analysis revealed that cyclosporine down-regulated 57 genes whose expression levels were increased by TGF-ß, and up-regulated 73 genes, whose expression was decreased by TGF-ß. Classifying these 57 down-regulated and 73 up-regulated genes with the Database for Annotation, Visualization and Integrated Discovery (DAVID) web tool, we have identified the involvement of several functional categories, including innate immunity, cytokine interaction, growth factor, and cancer pathway. Of the identified genes, we selected three fibrosis-related genes, insulin-like growth factor binding protein 2 (IGFBP2), inhibitor of DNA binding 1 (ID1) and peroxisome proliferator-activated receptor gamma (PPARG), and validated their expression patterns by quantitative reverse transcription-polymerase chain reaction. Cyclosporine treatment decreased the expression levels of IGFBP2 and ID1, but increased PPARG expression. These results suggest that cyclosporine is a potent anti-fibrotic agent acting on myofibroblasts. Therefore, cyclosporine shows potential as a novel remedy for fibrotic lung disease.

Zyxin modulates the transmigration of Haemophilus influenzae to the central nervous system.

The mechanism by which Haemophilus influenzae causes meningitis is unclear. Previously, we established murine meningitis by intranasal instillation of H. influenzae as a cell-bound organism (CBO). In this study, we aimed to identify the molecules associated with inhibiting the transmigration of cells across the blood-brain barrier (BBB). Two-dimensional difference gel electrophoresis and protein identification by mass spectrometry were used for proteomic analysis. Analysis of the membranous extract from a tumor necrosis factor (TNF)-α-treated human brain microvascular endothelial cell (HBMEC) monolayer revealed 41 differentially expressed proteins. Zyxin, which is thought to be essential for tight cell-to-cell junctions, decreased 1.8-fold in TNF-α-treated HBMECs. In addition, zyxin transcript levels decreased 1.5-fold in cells derived from TNF-α-treated HBMECs. Intranasal instillation of CBOs in zyxin-deficient mice resulted in a significant higher mortality rate than in wild-type mice. Transmigration of CBOs across a HBMEC monolayer pretreated with TNF-α (1 ng/mL), interleukin (IL)-1ß (10 ng/mL), or lipopolysaccharide (LPS; 10 ng/mL) was assayed by counting CBOs that migrated from an upper chamber into a lower chamber. HBMEC pretreated with TNF-α exhibited significantly greater migration (P<0.01) than did control cells or cells treated with IL-1ß or LPS. Our findings highlight that zyxin is an important protein protecting the tight junction of the BBB against cell transmigration across the BBB. Finally, TNF-α produced in respiratory infection when the primary infection reached the BBB caused decreased zyxin levels in BBB cell membranes. Furthermore, H. influenzae reaching the BBB as CBOs could transmigrate into cerebrospinal fluid across the zyxin-decreased BBB.

Polyphosphate-mediated inhibition of tartrate-resistant acid phosphatase and suppression of bone resorption of osteoclasts.

Inorganic polyphosphate (poly(P)) has recently been found to play an important role in bone formation. In this study, we found that tartrate-resistant acid phosphatase (TRAP), which is abundantly expressed in osteoclasts, has polyphosphatase activity that degrades poly(P) and yields Pi as well as shorter poly(P) chains. Since the TRAP protein that coprecipitated with anti-TRAP monoclonal antibodies exhibited both polyphosphatase and the original phosphatase activity, poly(P) degradation activity is dependent on TRAP and not on other contaminating enzymes. The ferrous chelator α, α'-bipyridyl, which inhibits the TRAP-mediated production of reactive oxygen species (ROS), had no effect on such poly(P) degradation, suggesting that the degradation is not dependent on ROS. In addition, shorter chain length poly(P) molecules were better substrates than longer chains for TRAP, and poly(P) inhibited the phosphatase activity of TRAP depending on its chain length. The IC50 of poly(P) against the original phosphatase activity of TRAP was 9.8 µM with an average chain length more than 300 phosphate residues, whereas the IC50 of poly(P) with a shorter average chain length of 15 phosphate residues was 8.3 mM. Finally, the pit formation activity of cultured rat osteoclasts differentiated by RANKL and M-CSF were markedly inhibited by poly(P), while no obvious decrease in cell number or differentiation efficiency was observed for poly(P). In particular, the inhibition of pit formation by long chain poly(P) with 300 phosphate residues was stronger than that of shorter chain poly(P). Thus, poly(P) may play an important regulatory role in osteoclastic bone resorption by inhibiting TRAP activity, which is dependent on its chain length.

Selection of autophagy or apoptosis in cells exposed to ER-stress depends on ATF4 expression pattern with or without CHOP expression.

Cells exposed to ER-stress undergo the Unfolded Protein Response (UPR) to avoid apoptosis, but may also activate autophagy. However, the signal for selection of one of these two protective responses is unknown. To clarify the key switch between autophagy and apoptosis, we examined the correlation of UPR-related signals with autophagy and/or apoptosis inductions in HepG2 cells exposed to three ER-stress inducers (NaF, tunicamycin, and thapsigargin) with time, including the effect of small interfering RNA on the cell responses. Thapsigargin-induced ER-stress caused only apoptosis after ∼2 hr with Ire1 phosphorylation, and Grp78, ATF4, and CHOP expressions. On the other hand, NaF- and tunicamycin-induced ER-stress caused only autophagy in the early stage by ∼8 hr with ATF4 expression and without CHOP expression. ATF4-siRNA completely inhibited the autophagy induced by NaF or tunicamycin with suppressed ATF4 protein and mRNA expressions, and also inhibited apoptosis by thapsigargin with suppression of both ATF4 and CHOP. CHOP-siRNA had no effect on autophagy activation by NaF and tunicamycin. On the other hand, CHOP-siRNA activated autophagy in thapsigargin-induced ER-stress with significant ATF4 expression, and suppressed apoptosis with CHOP suppression. These results showed that ATF4 is the key signal for autophagy induced by ER-stress, and that autophagy is switched to apoptosis by subsequent CHOP upregulation, suggesting that the changeover switch between autophagy and apoptosis is located between ATF4 to CHOP in the PERK pathway.

Sar1 translocation onto the ER-membrane for vesicle budding has different pathways for promotion and suppression of ER-to-Golgi transport mediated through H89-sensitive kinase and ER-resident G protein.

ER-to-Golgi protein transport involves transport vesicles of which formation is initiated by assembly of Sar1. The assembly of Sar1 is suppressed by protein kinase inhibitor H89, suggesting that ER-to-Golgi transport is regulated progressively by H89 sensitive kinase. ER-resident G(i2) protein suppresses vesicle formation with inhibition of Sar1 assembly. This study examined whether these promotion and suppression of vesicle transport share the same signal pathway, by examining the effects of G(i/o) protein activator mastoparan 7 (Mp-7) and H89 on Sar1 and Sec23 recruitment onto microsomes. In a cell-free system for Sar1 translocation assay, GTPγS addition induced the translocation of Sar1 onto microsomes. Mp-7 and H89 decreased the Sar1 translocation. Double treatment of Mp-7 and H89 strongly decreased Sar1 translocation. In single and double treatments, however, G(i/o) protein inactivator pertussis toxin (IAP) partially restored the suppressive effect of Mp-7, but had not any effect on H89-induced effect. Then, the assembly of Sec23 onto the microsome was also increased by the addition of GTPγS. Sec23 translocation was decreased by Mp-7 and/or H89 treatment and recovered by IAP pretreatment except for H89 single treatment, similarly to Sar1 translocation in each treatment. Inhibitory effects of H89 and Mp-7on ER-to-Golgi vesicle transport by H89 or Mp-7 were also confirmed in a cell culture system by BFA-dispersion and BFA-reconstruction experiments. These findings indicate that promotion and suppression of ER-to-Golgi vesicle transport are modulated through separate signal pathways.

ER-resident Gi2 protein controls sar1 translocation onto the ER during budding of transport vesicles.

In our previous study, fluoride ([AlF(4) ](-) ) disturbed ER-to-Golgi transport through the activation of ER-resident heterotrimeric G protein (ER-G protein). Therefore, ER-G protein may be implicated in ER-to-Golgi transport at the early stage prior to coat protein assembly. Sar1 translocation onto the endoplasmic reticulum (ER) membrane is suppressed by non-selective protein kinase inhibitor H89, suggesting the participation of H89-sensitive kinase in this process. To investigate the involvement of ER-G protein in ER-to-Golgi transport, the effect of G(i) protein activator (mastoparan 7) was examined on Sar1 translocation onto the ER in a cell-free system consisting of microsome membrane and cytosol. Sar1 translocation onto the microsome membrane was induced by addition of GTPγS in the cell-free system. Translocation of Sar1 by GTPγS was suppressed significantly by both H89 and mastoparan 7. Mastoparan 7 suppressed the translocation of Sar1 onto the microsome membrane with dosage dependency, but mastoparan 17, the inactive analog of mastoparan 7, had no effect on Sar1 translocation. The suppressive effect of mastoparan 7 was recovered by treatment with pertussis toxin (IAP). Moreover, G(i2) protein was detected on the microsome membrane by western blotting for heterotrimeric G(i) proteins. These results indicate that ER-G(i2) protein modulated Sar1 translocation onto the ER, suggesting that ER-resident G(i2) protein is an important negative regulator of vesicular transport at the early stage of vesicle formation before coat protein assembly on the ER.

H89 sensitive kinase regulates the translocation of Sar1 onto the ER membrane through phosphorylation of ER-coupled ß-tubulin.

ER-to-Golgi protein transport is carried out by transport vesicles which are formed at the ER-exit sites with recruitment of cytoplasmic coat proteins. Vesicle formation is initiated by assembly of the small G protein (Sar1) onto the ER membrane. Sar1 assembly onto the ER membrane is suppressed by protein kinase inhibitor H89, suggesting participation of H89-sensitive kinase in this process. The present study identified an effector of H89-sensitive kinase by LC-MS PMF analysis combined with 1D- and 2D-PAGE autoradiography, and examined the changes on the effector and Sar1 translocation induced by H89. H89 significantly suppressed the phosphorylation of 55 kDa protein with dosage dependency, and phosphorylation of 55 kDa, pI 5.5 protein spot in 2-D-autoradiography was drastically diminished by H89. LC-MS PMF analysis showed that the protein spot was ß-tubulin. H89 significantly suppressed Sar1 translocation onto the ER. These findings indicate that ß-tubulin is one of downstream effectors of H89-sensitive kinase, and that suppression of ER-coupled ß-tubulin phosphorylation decreases Sar1 translocation onto the ER, suggesting that phosphorylation of ß-tubulin regulates Sar1 translocation.

Effects of extracellular acidic-alkaline stresses on trigeminal ganglion neurons in the mouse embryo in vivo.

Acidic-alkaline stresses caused by ischemia and hypoglycemia induce neuronal cell death resulting from intracellular pH disturbance. The effects of acidic-alkaline disturbance on the trigeminal ganglion (TG) neurons of the embryonic mouse were investigated by caspase-3-immunohistochemistry and Nissl staining. TG neurons exhibited apoptosis in 3.08 ± 0.55% of neurons in intact embryos at day 16. Intraperitoneal injection of alkaline solution (pH 8.97; 0.005-0.1 M K2HPO4 or 0.01-0.04 M KOH) into the embryo at embryonic day 15 significantly increased the number of apoptotic neurons in the TG at embryonic day 16 with dependence on concentration (3.40-6.05 and 2.93-5.55%, respectively). On the other hand, acidic solutions (pH 4.4; 0.01-0.2 M KH2PO4 slightly, but not significantly, increased the number of apoptotic cells (3.64-5.15%, without dependence on concentration). Neutral solutions (pH 7.4; 0.01-0.2 M potassium phosphate buffer) had no effect on neuronal survival in the TG (2.89-3.48%). The results indicated that alkaline stress significantly increased apoptosis in the developing nervous system, but acidic stress did not.

New internalized distraction device for craniofacial plastic surgery using Ni-free, Ti-based shape memory alloy.

This study was undertaken to examine effects and biocompatibility of a new internalized distraction device made from newly developed Ti-Nb-Al shape memory alloy (SMA). Crania of Wistar rats were expanded using a U-shaped wire of this SMA set on each cranium in an experimental group. At 2 or 4 weeks after operation, the rats were killed; width measurements and three-dimensional observations of crania were conducted using soft x-ray and microfocus x-ray computed tomography photography. After photography, histologic sections were made and stained with hematoxylin and eosin. No pathologic change in the experimental duration was observed macroscopically or histologically. Significantly increased size was found for the rat crania in the experimental group compared with the control group. Results demonstrated the feasibility and biocompatibility of internalized distraction osteogenesis using Ni-free, Ti-based SMA in craniofacial plastic surgery for craniofacial deformities.

Development of an enzyme-linked immunosorbent assay for measurement of rat pulmonary surfactant protein D using monoclonal antibodies.

Surfactant protein D (SP-D) has been used as a biomarker of lung inflammation. In rat, several types of enzyme-linked immunosorbent assay (ELISA) using polyclonal antibodies have been reported. The purpose of this study was the development of a sensitive ELISA for rat SP-D using monoclonal antibodies. The authors developed a sandwich ELISA using monoclonal antibodies that were obtained by immunizing with purified rat SP-D. The ELISA was evaluated by performance tests. Furthermore, concentrations of serum SP-D were measured in normal control and bleomycin-treated rats. The working range of ELISA was between 0.47 and 30 ng/mL. Different concentrations of added SP-D were recovered, between 94.1% and 102.8%. Serum SP-D levels of bleomycin-treated rats were significantly higher than those of normal rats. In conclusion, this newly developed ELISA for rat SP-D using monoclonal antibodies is applicable for research on the mechanism and therapy of lung injury.

Involvement of endotoxin in the mortality of mice with gut-derived sepsis due to methicillin-resistant Staphylococcus aureus.

MRSA causes a wide diversity of diseases, ranging from benign skin infections to life-threatening diseases, such as sepsis. However, there have been few reports of the pathophysiology and mechanisms of sepsis resulting from the gut-derived origin of MRSA. Therefore, we established a murine model of gut-derived sepsis with MRSA and factors of MRSA sepsis that cause deterioration. We separated mice into four groups according to antibiotic treatment as follows: (i) ABPC 40 mg/kg; (ii) CAZ 80 mg/kg; (iii) CAZ 80 mg/kg + endotoxin 10 microg/mouse; and (iv) saline-treated control groups. Gut-derived sepsis was induced by i.p. injection of cyclophosphamide after colonization of MRSA strain 334 in the intestine. After the induction of sepsis, significantly more CAZ-treated mice survived compared with ABPC-treated and control groups. MRSA were detected in the blood and liver among all groups. Endotoxin levels were significantly lower in the CAZ-treated group compared to other groups. Inflammatory cytokine levels in the serum were lower in the CAZ-treated group compared to other groups. Fecal culture showed a lower level of colonization of E. coli in the CAZ-treated group compared to other groups. In conclusion, we found that CAZ-treatment ameliorates infection and suppresses endotoxin level by the elimination of E. coli from the intestinal tract of mice. However, giving endotoxin in the CAZ-treated group increased mortality to almost the same level as in the ABPC-treated group. These results suggest endotoxin released from resident E. coli in the intestine is involved in clinical deterioration resulting from gut-derived MRSA sepsis.