IRF3 polymorphisms induce different innate anti-Theiler's virus immune responses in RAW264.7 macrophages.

Persistent viral infections can lead to disease such as myocarditis. Theiler's murine encephalomyelitis virus (TMEV) infects macrophages of SJL/J (H-2s) mice establishing persistent infections leading to demyelinating disease. In contrast macrophages from B10.S (H-2s) mice clear TMEV. Activation of the transcription factor IRF3 induces IFNß, ISG56, and apoptosis for viral clearance, but also inflammatory cytokines, such as IL-23 and IL6, which contribute to disease. Here we identify polymorphisms in the IRF3 of SJL/J versus B10.S mice that are located in DNA binding, nuclear localization, and autoinhibitory domains. SJL-IRF3 expression in RAW264.7 macrophage cells with or without TMEV infection decreased IL-23p19 promoter activity compared with B10S-IRF3. In contrast SJL-IRF3 increased IL-6, ISG56 and IFNß in response to TMEV. B10S-IRF3 expression augmented apoptotic caspase activation and decreased viral RNA in TMEV-infected macrophages while SJL-IRF3 increased viral replication with less caspase activation. Therefore IRF3 polymorphisms contribute to viral persistence and altered cytokine expression.

Incidence of endodontic implantitis and implant endodontitis occurring with single-tooth implants: a retrospective study.

The purposes of this study were to determine success and survival rates for implants and teeth adjacent to implants and the incidence of endodontic implantitis (E-I) (endodontic involvement in adjacent teeth causing implant failure) and implant endodontitis (I-E) (implant placement causing endodontic failure). The data were from 233 single-tooth implants placed in 116 subjects by postgraduate periodontal students with recall radiographs taken >or=9 months after implant placement. Three groups were analyzed: group A, implants with no adjacent teeth (n = 90); group B, implants with nonendodontically treated adjacent teeth (n = 123); and group C, implants with endodontically treated adjacent teeth (n = 20). The success and survival rates for implants were both 92.2% in group A, 98.4% and 99.2% for group B, and 85% and 95% for group C, respectively. For adjacent teeth, they were both 99.4% in group B compared with 75% and 90% in group C. However, after case review, none of the implants or adjacent teeth in group B were considered to have E-I or I-E, and one (5%) of the implants in group C had E-I and two (10%) of the adjacent teeth may have had I-E. The results of the present study agree with previous research, which suggests that endodontically treated teeth adjacent to single-tooth implants are usually successful and should be maintained.

Promoter analysis reveals critical roles for SMAD-3 and ATF-2 in expression of IL-23 p19 in macrophages.

IL-23 p19/p40, produced by macrophages and dendritic cells, is critical for development of Th17 in several autoimmune diseases. In this study, bone marrow-derived (BMM) and splenic macrophages (SPM) from SJL/J mice, susceptible to autoimmune demyelinating disease following Theiler's virus (TMEV) infection, expressed IL-23 in response to TMEV. We identified potential binding sites for IFN response factor (IRF)-3 (nt -734 to -731), Sma- and Mad-related protein (SMAD)-3 (nt -584 to -581), activating transcription factor (ATF)-2 (nt -571 to -568), IRF-7 (nt -533 to-525), and NF-kappaB (nt -215 to -209) in the murine p19 promoter. The p19(prom) in the pGL3 promoter-reporter vector responded to TMEV or poly(I:C), a TLR3 agonist in the RAW264.7 macrophage cell line. Deletions upstream from the IRF-3 site and mutations at the IRF-3, SMAD-3, ATF-2, or NF-kappaB, but not the IRF-7, sites significantly reduced promoter activity. ATF-2 or SMAD-3, but not IRF-3, short-hairpin RNA reduced p19 promoter activity and protein expression in RAW264.7 cells responding to TMEV. Chromosomal DNA immunoprecipitation assays revealed that SMAD-3 and ATF-2 bind to the endogenous p19 promoter in RAW264.7 cells and SJL/J SPM following challenge with TMEV. TGF-beta1, which activates SMAD-3, was induced in RAW264.7 cells, BMM, and SPM by TMEV. Neutralizing Ab to TGF-beta1 eliminated TMEV-induced IL-23 production and SMAD-3 activation in RAW264.7 cells, BMM, and SPM. Activation of ATF-2 was JNK, but not p38 or ERK MAPK dependent. Inhibition of the JNK, but also the ERK MAPK pathways decreased expression of p19. These results suggest that ATF-2 and SMAD-3 are transcription factors, which are, in addition to NF-kappaB, essential for IL-23 p19 expression.

Human osteogenic protein-1 induces osteogenic differentiation of adipose-derived stem cells harvested from mice.

OBJECTIVE: Osteogenic protein-1 (OP-1) has been shown to stimulate undifferentiated cells to produce mineralized tissue. Adipose tissue is a rich source of undifferentiated cells for tissue engineering purposes. The purpose of this study was to investigate the effect of OP-1 on osteogenic differentiation of adipose-derived stem cells and the production of bony tissue in vitro. DESIGN: Adipose-derived stem cells (ADSCs) were isolated from inguinal fat pads of adult mice. Following cell expansion the cells were plated in 8-well chambered slides. The cells received one of four treatments: Group 1 cells were maintained in control medium, Group 2 cells were cultured in a common osteogenic medium, Group 3 cells were cultured in osteogenic medium supplemented with 250ng/mL of OP-1, and Group 4 cells were cultured with 250ng/mL of OP-1 added to control medium. Osteogenic differentiation of ADSCs was determined by estimating the number and size of mineralized nodules, and the amount of extracellular osteopontin secreted into cell culture medium. Mineralized nodule production was assessed at day 21 with von Kossa staining. Extracellular osteopontin release was measured after 8 and 21 days by enzyme-linked immunosorbant assay (ELISA). ANOVA/Tukey tests were used to identify differences among the four treatment groups for mineralized nodule production and osteopontin release (p0.05), which were significantly higher than the group incubated in cell growth medium only (p0.05). Linear regression analysis demonstrated a linear relationship was present between the presence of calcified nodules and the amount of osteopontin released (p

TLR3 and TLR7 are involved in expression of IL-23 subunits while TLR3 but not TLR7 is involved in expression of IFN-beta by Theiler's virus-infected RAW264.7 cells.

Theiler's murine encephalomyelitis virus (TMEV) infects macrophages and causes demyelinating disease (DD) in certain mouse strains. IL-23 p19/p40 and IFN-beta, which are both expressed by macrophages in response to TMEV, could contribute to or prevent DD. Because TMEV may induce macrophages' cytokines through TLR3 and TLR7 (toll-like receptors), their role in TMEV-induced IL-23 and IFN-beta expression by the RAW264.7 macrophage cell line was determined following infection with TMEV or stimulation with the poly (I:C) or loxoribine. TMEV infection or stimulation with poly (I:C), a TLR3 agonist, or loxoribine, a TLR7 agonist, induced expression of IL-23 and IFN-beta in RAW264.7 cells. In addition, TMEV infection increased expression of TLR3 and TLR7 in RAW264.7 cells. Transfection of RAW264.7 cells with shRNA plasmid vectors expressing siRNA specific for TLR3 or TLR7 concomitantly decreased expression of TLR3 or TLR7, respectively, and TMEV-induced p19 mRNA, p19 protein, and IL-23 p19/p40. Transfection with TLR7-shRNA plasmids reduced expression of TMEV-induced p40 mRNA and p40 protein. However, transfection with TLR3-shRNA plasmids increased expression of TMEV-induced p40 mRNA but decreased p40 protein. In addition, transfection with TLR3-shRNA plasmids but not TLR7-shRNA plasmids decreased expression of TMEV-induced IFN-beta mRNA. Thus TLR3 and TLR7 contribute to TMEV-induced IL-23 p19 and p40, while TLR3 contributes to TMEV-induced IFN-beta.

Expression of IL-27 p28 by Theiler's virus-infected macrophages depends on TLR3 and TLR7 activation of JNK-MAP-kinases.

Theiler's murine encephalomyelitis virus (TMEV) causes a demyelinating disease (DD) due to infection of macrophages, stimulation of macrophage Toll-like receptor (TLR)3 and TLR7 pathways, activation of Mitogen-activated protein kinases (MAPK)s, and production of macrophages cytokines. Because expression of IL-27, a macrophage cytokine composed of p28 and EBI3 subunits, has been implicated in DD, we examined IL-27 subunit mRNA expression during TMEV infection of RAW264.7 cells, a macrophage cell line. TMEV infection of RAW264.7 cells did not affect cell viability, resulted in viral RNA replication, as well as p28 and EBI3 expression. Expression of p28 in TMEV-infected RAW264.7 cells depended on TLR3 and TLR7, as well as JNK but not p38 or ERK MAPKs. Since TMEV causes DD in SJL/J but not B10.S mice we determined the difference in expression of IL-27 subunit mRNA in SJL/J compared to B10.S macrophages. SJL/J macrophages expressed significantly more p28 mRNA after TMEV infection and after stimulation with TLR3 and TLR7 agonists compared with B10.S macrophages. Therefore, macrophages expression of IL-27 p28 mRNA in response to TMEV is due to activation of TLR3, TLR7, and JNK MAPKs pathways.