Activation of NLRP3 Inflammasome Complexes by Beta-Tricalcium Phosphate Particles and Stimulation of Immune Cell Migration in vivo.

Beta-tricalcium phosphate (ß-TCP) serves as a bone substitute in clinical practice because it is resorbable, biocompatible, osteointegrative, and osteoconductive. Particles of ß-TCP are also inflammatory mediators although the mechanism of this function has not been fully elucidated. Regardless, the ability of ß-TCP to stimulate the immune system might be useful for immunomodulation. The present study aimed to determine the effects of ß-TCP particles on NLR family pyrin domain containing 3 (NLRP3) inflammasome complexes. We found that ß-TCP activates NLRP3 inflammasomes, and increases interleukin (IL)-1ß production in primary cultured mouse dendritic cells (DCs) and macrophages, and human THP-1 cells in caspase-1 dependent manner. In THP-1 cells, ß-TCP increased also IL-18 production, and NLRP3 inflammasome activation by ß-TCP depended on phagocytosis, potassium efflux, and reactive oxygen species (ROS) generation. We also investigated the effects of ß-TCP in wild-type and NLRP3-deficient mice in vivo. Immune cell migration around subcutaneously injected ß-TCP particles was reduced in NLRP3-deficient mice. These findings suggest that the effects of ß-TCP particles in vivo are at least partly mediated by NLRP3 inflammasome complexes.

Effects of beta-tricalcium phosphate particles on primary cultured murine dendritic cells and macrophages.

Beta-tricalcium phosphate (ß-TCP) is widely used for bone substitution in clinical practice. Particles of calcium phosphate ceramics including ß-TCP act as an inflammation mediators, which is an unfavorable characteristic for a bone substituent or a prosthetic coating material. It is thought that the stimulatory effect of ß-TCP on the immune system could be utilized as an immunomodulator. Here, in vitro effects of ß-TCP on primary cultured murine dendritic cells (DCs) and macrophages were investigated. ß-TCP particles enhanced expression of costimulatory surface molecules, including CD86, CD80, and CD40 in DCs, CD86 in macrophages, and MHC class II and class I molecules in DCs. DEC205 and CCR7 were up-regulated in ß-TCP-treated DCs. Production of cytokines and chemokines, including CCL2, CCL3, CXCL2, and M-CSF, significantly increased in DCs; CCL2, CCL3, CCL4, CCL5, CXCL2, and IL-11ra were up-regulated in macrophages. The results of the functional assays revealed that ß-TCP caused a prominent reduction in antigen uptake by DCs, and that conditioned medium from DCs treated with ß-TCP facilitated the migration of splenocytes in the transwell migration assay. Thus, ß-TCP induced phenotypical and functional maturation/activation of DCs and macrophages; these stimulating effects may contribute to the observed in vivo effect where ß-TCP induced extensive migration of immune cells. When compared to lipopolysaccharide (LPS), an authentic TLR ligand, the stimulatory effect of ß-TCP on the immune systems is mild to moderate; however, it may have some advantages as a novel immunomodulator. This is the first report on the direct in vitro effects of ß-TCP against bone marrow-derived DCs and macrophages.

Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of nine local anesthetic drugs.

A high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS) with electrospray ionization (ESI) procedure for the simultaneous determination of nine local anesthetic drugs (procaine, mepivacaine, lidocaine, ropivacaine, oxybuprocaine, tetracaine, bupivacaine, T-caine and dibucaine) in human serum is described. The chromatographic separation was performed on a Mightysil-RP-18 GP II column (2.0mm×150mm, particle size 5µm). The mobile phase consisted of 10mM acetic ammonium buffer (pH 5.4) and acetonitrile and was delivered at a flow rate of 0.20mL/min. The triple quadrupole mass spectrometer was operated in positive ion mode, and multiple reaction monitoring was used for drug quantification. Solid-phase extraction of the nine local anesthetic drugs added to the human serum was performed with an Oasis(®) HLB extraction cartridges column. The method was linear for the investigated drugs over the concentration range of 10-100ng/mL. The recoveries of these drugs were in the range of 81.4-144%. The standard deviation (SD) values for all analytes were <0.10 for both intraday and interday accuracy and precision. The selectivity, accuracy and precision of this method are satisfactory for clinical and forensic applications. The sensitive and selective method offers the opportunity for the simultaneous screening and quantification, for clinical and forensic purposes, of almost all local anesthetics available in Japan.

AW551984: a novel regulator of cardiomyogenesis in pluripotent embryonic cells.

An understanding of the mechanism that regulates the cardiac differentiation of pluripotent stem cells is necessary for the effective generation and expansion of cardiomyocytes as cell therapy products. In the present study, we have identified genes that modulate the cardiac differentiation of pluripotent embryonic cells. We isolated P19CL6 cell sublines that possess distinct properties in cardiomyogenesis and extracted 24 CMR (cardiomyogenesis-related candidate) genes correlated with cardiomyogenesis using a transcriptome analysis. Knockdown of the CMR genes by RNAi (RNA interference) revealed that 18 genes influence spontaneous contraction or transcript levels of cardiac marker genes in EC (embryonal carcinoma) cells. We also performed knockdown of the CMR genes in mouse ES (embryonic stem) cells and induced in vitro cardiac differentiation. Three CMR genes, AW551984, 2810405K02Rik (RIKEN cDNA 2810405K02 gene) and Cd302 (CD302 antigen), modulated the cardiac differentiation of both EC cells and ES cells. Depletion of AW551984 attenuated the expression of the early cardiac transcription factor Nkx2.5 (NK2 transcription factor related locus 5) without affecting transcript levels of pluripotency and early mesoderm marker genes during ES cell differentiation. Activation of Wnt/ß-catenin signalling enhanced the expression of both AW551984 and Nkx2.5 in ES cells during embryoid body formation. Our findings indicate that AW551984 is a novel regulator of cardiomyogenesis from pluripotent embryonic cells, which links Wnt/ß-catenin signalling to Nkx2.5 expression.

Antiviral activities against herpes simplex virus type 1 by HPH derivatives and their structure-activity relationships.

The compound named Histidine-pyridine-histidine (HPH) is an oxygen-activating ligand derived from the structure of bleomycin. We synthesized HPH derivatives, namely HPH-1 to -8, and investigated their antiviral activities against herpes simplex virus type 1. HPH-8 showed potent antiviral activity with an EC50 of 15 microM, and relatively high cytotoxicity with a CC50 of 37 microM. In contrast, HPH-4 indicated a weaker antiviral activity with an EC50 of 79 microM, but exhibited a far more less cytotoxicity (CC50 500 microM). Other HPH derivatives showed no effects against antiviral activities and cytotoxicities.

Rapid construction of small interfering RNA-expressing adenoviral vectors on the basis of direct cloning of short hairpin RNA-coding DNAs.

In the conventional method for constructing an adenoviral (Ad) vector expressing small interfering RNA (siRNA), short hairpin RNA (shRNA)-coding oligonucleotides are introduced downstream of a polymerase III (or polymerase II)-based promoter cloned into a shuttle plasmid. An siRNA expression cassette, which is cloned into the shuttle plasmid, is then introduced into the E1 deletion region of the Ad vector plasmid by in vitro ligation or homologous recombination in Escherichia coli, and the linearized plasmid is transfected into 293 cells, generating an Ad vector expressing siRNA. Therefore, two-step plasmid manipulation is required. In this study, we developed a method by which shRNA-coding oligonucleotides can be introduced directly into the Ad vector plasmid. To do this, we constructed a new vector plasmid into which the human U6 promoter sequence was cloned in advance. Unique restriction enzyme sites were introduced at the transcription start site of the U6 promoter sequence in the vector plasmid. Luciferase and p53 genes were efficiently knocked down by Ad vectors generated by the new method and expressing siRNA against the target gene. This method should be useful for RNA interference-based experiments, and should make it easy to construct an siRNA-expressing Ad vector library for functional screening.

Approaches to improving the kinetics of adenovirus-delivered genes and gene products.

Adenovirus (Ad) vectors have been expected to play a great role in gene therapy because of their extremely high transduction efficiency and wide tropism. However, due to the intrinsic deficiency of their immunogenic toxicities, Ad vectors are rapidly cleared from the host, transgene expression is transient, and readministration of the same serotype Ad vectors is problematic. As a result, Ad vectors are continually undergoing refinement to realize their potential for gene therapy application. Even after 1999, when a patient fatally succumbed to the toxicity associated with Ad vector administration at a University of Pennsylvania (U.S.) experimental clinic, enthusiasm of gene therapists for Ad vectors has not waned. With great efforts from various research groups, significant advances have been achieved through comprehensive approaches to improving the kinetics of Ad vector-delivered genes and gene products.

RNA interference of PPARgamma using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells.

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.

Adenovirus vector-mediated doxycycline-inducible RNA interference.

RNA interference (RNAi) is a powerful tool for the knockdown of gene expression. Here, we report on the development of an adenovirus (Ad) vector-mediated doxycycline (Dox)-inducible small interfering RNA (siRNA) expression system. We used this siRNA system to control the expression of p53 and c-Myc in human cancer cells. Coinfection of Ad vectors containing the siRNA expression system under the control of the Dox-inducible H1 promoter and Ad vectors expressing a tetracycline repressor inhibited the expression levels of p53 and c-Myc in a dose-dependent manner with both Dox and viral dose. Regulated silencing of p53 and c-Myc expression was obtained. Because an Ad vector-mediated inducible RNAi system can efficiently transduce a variety of cell types in vitro and in vivo, and the degree of loss of gene expression can be modulated according to the dose of Dox, this expression system should be a useful tool for both basic research on the analysis of gene function and therapeutic applications of RNAi.