Evaluation of New Octacalcium Phosphate-Coated Xenograft in Rats Calvarial Defect Model on Bone Regeneration.

Bone graft material is essential for satisfactory and sufficient bone growth which leads to a successful implant procedure. It is classified into autogenous bone, allobone, xenobone and alloplastic materials. Among them, it has been reported that heterogeneous bone graft material has a porous microstructure that increases blood vessels and bone formation, and shows faster bone formation than other types of bone graft materials. We observed new bone tissue formation and bone remodeling using Ti-oss® (Chiyewon Co., Ltd., Guri, Korea), a heterologous bone graft material. Using a Sprague-Dawley rat calvarial defect model to evaluate the bone healing effect of biomaterials, the efficacy of the newly developed xenograft Ti-oss® and Bio-Oss® (Geistilch Pharma AG, Wolhusen, Switzerland). The experimental animals were sacrificed at 8 and 12 weeks after surgery for each group and the experimental site was extracted. The average new bone area for the Ti-oss® experimental group at 8 weeks was 17.6%. The remaining graft material was 22.7% for the experimental group. The average new bone area for the Ti-oss® group was 24.3% at 12 weeks. The remaining graft material was 22.8% for the experimental group. It can be evaluated that the new bone-forming ability of Ti-oss® with octacalcium phosphate (OCP) has the bone-forming ability corresponding to the conventional products.

Effect of Lactobacillus rhamnosus GR-1 Supernatant on Cytokine and Chemokine Output From Human Amnion Cells Treated With Lipoteichoic Acid and Lipopolysaccharide.

Preterm birth occurs in 9% to 13% of all human pregnancies and accounts for 80% of all neonatal morbidities and mortalities. Approximately 40% of all preterm births are idiopathic and about half are associated with infection and/or an activated inflammatory process. Further to studies showing anti-inflammatory effects of supernatant from the probiotic Lactobacillus rhamnosus GR-1 (GR-1), we tested its ability to modulate cytokine and chemokine production from amnion cells in response to stimulation by bacterial wall components, lipopolysaccharide (LPS), and lipoteichoic acid (LTA). Placentae were collected from women undergoing elective cesarean section at term. Amnion cells were cultured for 48 hours to confluence, serum starved for 12 hours, and then treated with GR-1 supernatant (1:20 dilution), followed after 12 hours by LPS (100 ng/mL) or LTA (10 ng/mL) for an additional 12 hours. Both LTA and LPS caused significant increases in the concentration of the pro-inflammatory cytokine, tumor necrosis factor α (TNF-α; 103.9 ± 67.5 pg/mL and 368.3 ± 65.7 pg/mL, respectively) in medium from cultured amnion cells compared to control (<4 pg/mL). There was no significant effect of GR-1 supernatant alone on TNF-α output, but there was significant reduction after LPS treatment. The basal output of the immunomodulatory cytokine, interleukin 6, was 613 ± 170 pg/mL and increased significantly after addition of GR-1 supernatant, LTA, LPS, and combinations of LTA/LPS with GR-1 supernatant. In conclusion, probiotic L rhamnosus GR-1 attenuates the effect of both LPS and LTA in stimulating the output of the pro-inflammatory cytokine TNF-α from mixed cultures of human amnion cells in keeping with previous findings in human trophoblast cells.

Probiotic Lactobacillus rhamnosus GR-1 supernatant prevents lipopolysaccharide-induced preterm birth and reduces inflammation in pregnant CD-1 mice.

OBJECTIVE: The objective of this study was to determine the effect of probiotic Lactobacillus rhamnosus GR-1 supernatant (GR-1 SN) on lipopolysaccharide-induced preterm birth (PTB) and outputs of cytokines, chemokines, and progesterone in pregnant CD-1 mice. STUDY DESIGN: We compared PTB rates after intrauterine injection of lipopolysaccharide with and without previous GR-1 SN treatment. Cytokines and chemokines in the maternal plasma, myometrium, placenta, and amniotic fluid were examined with multiplex assay, and circulating maternal progesterone was measured with enzyme-linked immunoassay. Statistical significance was assessed with 2-tailed 1-way analysis of variance or analysis of variance on ranks. Fetal sex ratios in mice that delivered preterm were compared with those that delivered at term after lipopolysaccharide and GR-1 SN treatments. RESULTS: GR-1 SN reduced lipopolysaccharide-induced PTB by 43%. GR-1 SN significantly decreased the lipopolysaccharide-induced production of interleukin (IL)-1ß, -6, and -12p40, tumor necrosis factor-α, CCL4, and CCL5 in maternal plasma; IL-6, -12p70, -17, and -13 and tumor necrosis factor-α in myometrium; IL-6, -12p70, and -17 in placenta; and IL-6, tumor necrosis factor-α, CCL3, and CCL4 in amniotic fluid. Maternal plasma progesterone was reduced significantly after lipopolysaccharide injection with and without GR-1 SN pretreatment. There was no difference in fetal sex ratios between mice that delivered preterm and those that did not after lipopolysaccharide and GR-1 SN treatments. CONCLUSION: The supernatant of probiotic L rhamnosus GR-1 attenuated lipopolysaccharide-induced inflammation and PTB in vivo. GR-1 SN may confer therapeutic benefits in the prevention of infection-associated PTB by controlling systemic and intrauterine inflammation.

Lipopolysaccharide-Induced Profiles of Cytokine, Chemokine, and Growth Factors Produced by Human Decidual Cells Are Altered by Lactobacillus rhamnosus GR-1 Supernatant.

The aim of this study was to assess the effects of bacterial lipopolysaccharide (LPS) and Lactobacillus rhamnosus GR-1 supernatant (GR-1SN) on secretion profiles of cytokines, chemokines, and growth factors from primary cultures of human decidual cells. Lipopolysaccharide significantly increased the output of proinflammatory cytokines (interleukin [IL]-1B, IL-2, IL-6, IL-12p70, IL-15, IL-17A, interferon gamma [IFN-γ], and tumor necrosis factor [TNF]); anti-inflammatory cytokines (IL-1RN, IL-4, IL-9, and IL-10); chemokines (IL-8, eotaxin, IFN-inducible protein 10 [IP-10], monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein-1α [MIP-1α], macrophage inflammatory protein-1ß [MIP-1ß], and regulated on activation normal T cell expressed and secreted [RANTES]); and growth factors (granulocyte colony-stimulating factor [CSF] 3, CSF-2, and vascular endothelial growth factor A [VEGFA]). Lactobacillus rhamnosus GR-1SN alone significantly increased CSF-3, MIP-1α MIP-1ß, and RANTES but decreased IL-15 and IP-10 output. The GR-1SN also significantly or partially reduced LPS-induced proinflammatory cytokines TNF, IFN-γ, IL-1ß, IL-2 IL-6, IL-12p70, IL-15, IL-17, and IP-10; partially reduced LPS-induced anti-inflammatory cytokines IL-1RN, IL-4 and IL-10, and LPS-induced VEGFA output but did not affect CSF-3, MIP-1α, MIP-1ß, MCP-1, IL-8, and IL-9. Our results demonstrate that GR-1SN attenuates the inflammatory responses to LPS by human decidual cells, suggesting its potential role in ameliorating intrauterine infection.

Role of parathyroid hormone-related protein in the pro-inflammatory and pro-fibrogenic response associated with acute pancreatitis.

Pancreatitis is a common and potentially lethal necro-inflammatory disease with both acute and chronic manifestations. Current evidence suggests that the accumulated damage incurred during repeated bouts of acute pancreatitis (AP) can lead to chronic disease, which is associated with an increased risk of pancreatic cancer. While parathyroid hormone-related protein (PTHrP) exerts multiple effects in normal physiology and disease states, its function in pancreatitis has not been previously addressed. Here we show that PTHrP levels are transiently elevated in a mouse model of cerulein-induced AP. Treatment with alcohol, a risk factor for both AP and chronic pancreatitis (CP), also increases PTHrP levels. These effects of cerulein and ethanol are evident in isolated primary acinar and stellate cells, as well as in the immortalized acinar and stellate cell lines AR42J and irPSCc3, respectively. Ethanol sensitizes acinar and stellate cells to the PTHrP-modulating effects of cerulein. Treatment of acinar cells with PTHrP (1-36) increases expression of the inflammatory mediators interleukin-6 (IL-6) and intracellular adhesion protein (ICAM-1), suggesting a potential autocrine loop. PTHrP also increases apoptosis in AR42J cells. Stellate cells mediate the fibrogenic response associated with pancreatitis; PTHrP (1-36) increases procollagen I and fibronectin mRNA levels in both primary and immortalized stellate cells. The effects of cerulein and ethanol on levels of IL-6 and procollagen I are suppressed by the PTH1R antagonist, PTHrP (7-34). Together these studies identify PTHrP as a potential mediator of the inflammatory and fibrogenic responses associated with alcoholic pancreatitis.

Apoptosis of lung carcinoma cells induced by a flexible optical fiber-based cold microplasma.

Atmospheric pressure plasmas have been used as a therapy for cancer. However, the fairly large size and rigidity of present plasma-delivery systems obstructs the precise treatment of tumors in harder-to-reach internal organs such as the lungs, pancreas, and duodenum. In order to improve the targeted delivery of plasmas a highly flexible microplasma jet device is fabricated using a hollow-core optical fiber with an inner diameter of either 15 µm, 55 µm, or 200 µm. Described herein, based on this device, are results on lung carcinoma therapy using a microplasma cancer endoscope. Despite the small inner diameter and the low gas flow rate, the generated plasma jets are shown to be sufficiently effective to induce apoptosis, but not necrosis, in both cultured mouse lung carcinoma and fibroblast cells. Further, the lung carcinoma cells were found to be more sensitive to plasma treatment than the fibroblast cells based on the overall plasma dose conditions. This work enables directed cancer therapies using on highly flexible and precise hollow optical fiber-based plasma device and offers enhancements to microplasma cancer endoscopy using an improved method of plasma targeting and delivery.

Single-cell-level microplasma cancer therapy.

A flexible microplasma endoscope based on a 15 µm hollow-core glass optical fiber is fabricated, and tumor cell apoptotic analysis supports its potential use in targeted cancer therapies. The optical-fiber microplasma jet reveals antitumor activity at a certain plasma dose in animal studies.

Lactobacillus rhamnosus GR-1 stimulates colony-stimulating factor 3 (granulocyte) (CSF3) output in placental trophoblast cells in a fetal sex-dependent manner.

Bacterial vaginosis is associated with a 1.4-fold increased risk of preterm birth. We have shown previously that Lactobacillus rhamnosus GR-1 supernatant up-regulates interleukin 10 and down-regulates tumor necrosis factor-alpha output in lipopolysaccharide (LPS)-treated human primary placenta cultures in a fetal sex-dependent manner. We hypothesize that lactobacilli also exert their anti-inflammatory effect by up-regulation of colony-stimulating factor 3 (granulocyte) (CSF3), which is secreted from both immune and placental trophoblast cells, and that this activity is dependent on the sex of the fetus. Placental trophoblast cells were isolated from term elective cesarean section placentae using a Percoll gradient and separated from CD45(+) cells using magnetic purification. Cells were treated with LPS in the presence or absence of pretreatments with L. rhamnosus GR-1 supernatant or chemical inhibitors of the intracellular signaling pathways. Phosphorylations of mitogen-activated protein kinase 14 (MAPK14, previously known as p38) and signal transducer and activator of transcription (STAT) 3 were measured by Western blot analysis, and levels of CSF3 were determined by ELISA. CSF3 output was increased only in the placental trophoblast cells of female fetuses treated with LPS, GR-1 supernatant, and a combination of both treatments. The GR-1 supernatant up-regulated the phosphorylation of STAT3 and MAPK14. CSF3 output was inhibited by both Janus kinases (JAK) and MAPK14 inhibitors. None of the treatments was able to increase CSF3 output in either the pure trophoblast or the CD45(+) cell preparations alone. These results suggest an underlying mechanism for the sex difference in incidence of preterm birth and provide potential evidence for a therapeutic benefit of lactobacilli in reducing the risk of preterm labor.

15-µm-sized single-cellular-level and cell-manipulatable microplasma jet in cancer therapies.

The authors describe a proposed 15-µm-sized, single-cellular-level, and cell-manipulatable microplasma jet device with a microcapillary glass tip and its potential in the development of cancer treatment therapies. The electrical and optical properties of the plasma jets and preliminary apoptosis results of cultured murine tumor cells and non-tumor fibroblast cells treated with the plasma jets are presented. The generated plasma jet was stable and enabled the treatment of cultured cells in cell culture plates regardless of the small inner diameter and low gas flow rate. The microplasma jet was observed inducing apoptosis in cultured murine melanoma tumor cells in a dose-dependent manner. Furthermore, the percentage of apoptotic cells of murine melanoma tumor cells induced by this plasma device was approximately 2.5 times bigger than that of murine fibroblast cells as indicated by an Annex V apoptosis assay. The apoptosis in cultured murine tumor cells by the 15-µm-sized single-cellular-level and cell-manipulatable microplasma jet device was also observed using an in situ apoptosis assay. We report on a novel microplasma jet device with the advantages of single-cellular-level and single cell-manipulatable plasma treatment with precise and solid stimuli. This highly precise plasma medicine, which enables new directed cancer therapies can be combined with current cell manipulation and cell culturing technologies without much difficulty.

Effect of Lactobacillus rhamnosus GR-1 supernatant and fetal sex on lipopolysaccharide-induced cytokine and prostaglandin-regulating enzymes in human placental trophoblast cells: implications for treatment of bacterial vaginosis and prevention of preterm labor.

OBJECTIVE: The objective of the study was to determine the effect of fetal sex on the output of cytokines and prostaglandin-regulating enzymes in lipopolysaccharide (LPS) and probiotic lactobacilli-treated placental trophoblast cells. STUDY DESIGN: We examined the effect of LPS and Lactobacillus rhamnosus GR-1 supernatant in placental trophoblast cells on tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and IL-10 using enzyme-linked immunosorbent assay and on prostaglandin-endoperoxide synthase 2 (PTGS2), 15-hydroxy prostaglandin dehydrogenase (PGDH), and toll-like receptor-4 (TLR-4) using Western blotting. Comparisons were performed using one-way analysis of variance and Student t test. RESULTS: LPS increased the output of TNF-alpha, IL-10, and PTGS2 with a greater response in male placentae. L rhamnosus GR-1 supernatant inhibited the LPS-stimulated TNF-alpha and increased IL-10. It also up-regulated expression of PGDH in female placentae and partially reduced the LPS-stimulated PTGS2 in male placentae. There was no change in IL-1beta. Expression of TLR-4 was greater in placentae of male fetuses. CONCLUSION: These findings suggest an underlying mechanism for the sex difference in the incidence of preterm birth and provide potential evidence for a therapeutic benefit of lactobacilli in reducing preterm labor.

Mutagenesis by retroviral insertion in chemical mutagen-generated quasi-haploid mammalian cells.

Diploidy is a major obstacle to the mutagenic analysis of function in cultured mammalian cells. Here, we show that 6-8 rounds of chemical mutagenesis generates quasi-haploid cells that can be used as targets for insertional mutagenesis using a specially designed retroviral vector that permits rapid identification of disrupted genes in each cell that bears a phenotype of interest. The utility of combined chemical and insertional mutagenesis is illustrated by the identification of novel host genes that are required for macrophage sensitivity to anthrax lethal factor.

p85alpha acts as a novel signal transducer for mediation of cellular apoptotic response to UV radiation.

Apoptosis is an important cellular response to UV radiation (UVR), but the corresponding mechanisms remain largely unknown. Here we report that the p85alpha regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) exerted a proapoptotic role in response to UVR through the induction of tumor necrosis factor alpha (TNF-alpha) gene expression. This special effect of p85alpha was unrelated to the PI-3K-dependent signaling pathway. Further evidence demonstrated that the inducible transcription factor NFAT3 was the major downstream target of p85alpha for the mediation of UVR-induced apoptosis and TNF-alpha gene transcription. p85alpha regulated UVR-induced NFAT3 activation by modulation of its nuclear translocation and DNA binding and the relevant transcriptional activities. Gel shift assays and site-directed mutagenesis allowed the identification of two regions in the TNF-alpha gene promoter that served as the NFAT3 recognition sequences. Chromatin immunoprecipitation assays further confirmed that the recruitment of NFAT3 to the endogenous TNF-alpha promoter was regulated by p85alpha upon UVR exposure. Finally, the knockdown of the NFAT3 level by its specific small interfering RNA decreased UVR-induced TNF-alpha gene transcription and cell apoptosis. The knockdown of endogenous p85alpha blocked NFAT activity and TNF-alpha gene transcription, as well as cell apoptosis. Thus, we demonstrated p85alpha-associated but PI-3K-independent cell death in response to UVR and identified a novel p85alpha/NFAT3/TNF-alpha signaling pathway for the mediation of cellular apoptotic responses under certain stress conditions such as UVR.

G-CSF-mediated inhibition of JNK is a key mechanism for Lactobacillus rhamnosus-induced suppression of TNF production in macrophages.

Lactobacillus rhamnosus is a human commensal with known immunomodulatory properties. To date the mechanism of these immunomodulatory effects is not well understood. To unravel the immunomodulatory signalling mechanism, we investigated the effects of two strains of L. rhamnosus, L. rhamnosus GG and GR-1, in modulating production of tumour necrosis factor-alpha (TNF) in human monocytic cell line THP-1 and mouse macrophages. Live L. rhamnosus GG and GR-1 or their spent culture supernatant induced minuscule amounts of TNF production but large quantities of granulocyte-colony stimulating factor (G-CSF) in macrophages compared with those induced by pathogenic Escherichia coli GR-12 and Enterococcus faecalis. By using neutralizing antibodies and G-CSF receptor knockout mice, we demonstrated that G-CSF secreted from L. rhamnosus GG- and GR-1-exposed macrophages suppressed TNF production induced by E. coli- or lipopolysaccharide-activated macrophages through a paracrine route. The suppression of TNF production by G-CSF was mediated through activation of STAT3 and subsequent inhibition of c-Jun-N-terminal kinases (JNKs). The inhibition of JNK activation required STAT3alpha-mediated de novo protein synthesis. This demonstrates a novel role of G-CSF in L. rhamnosus-triggered anti-inflammatory effects and its mechanism in the suppression of TNF production in macrophages.

Upregulation of costimulatory molecules induced by lipopolysaccharide and double-stranded RNA occurs by Trif-dependent and Trif-independent pathways.

Both lipopolysaccharide (LPS) and double-stranded RNA (dsRNA) are adjuvants for the adaptive immune response, inducing upregulation of costimulatory molecules (UCM) on antigen-presenting cells. Trif, an adapter protein that transduces signals from Toll-like receptor 4 (TLR4) and TLR3, permits the induction of many cytokines, including interferon-beta, which signals through the type I interferon receptor. We show here that LPS-induced UCM was strictly dependent on the TLR4-->Trif axis, whereas dsRNA-induced UCM was only partly dependent on the TLR3-->Trif axis. But both LPS- and dsRNA-induced UCM were entirely dependent on type I interferon receptor signaling. These findings show that UCM involves an autocrine or paracrine loop, and indicate that an alternative TLR3-independent, Trif-independent pathway contributes to dsRNA-induced UCM.

Susceptibility of lysosomes to rupture is a determinant for plasma membrane disruption in tumor necrosis factor alpha-induced cell death.

Since a release of intracellular contents can induce local inflammatory responses, mechanisms that lead to loss of plasma membrane integrity in cell death are important to know. We showed previously that deficiency of the plasma membrane Ca2+ ATPase 4 (PMCA4) in L929 cells impaired tumor necrosis factor alpha (TNF-alpha)-induced enlargement of lysosomes and reduced cell death. The lysosomal changes can be determined by measuring the total volume of intracellular acidic compartments per cell (VAC), and we show here that inhibition of the increase in VAC due to PMCA4 deficiency not only reduced cell death but also converted TNF-alpha-induced cell death from a process involving disruption of the plasma membrane to a cell demise with a nearly intact plasma membrane. The importance of the size of lysosomes in determining plasma membrane integrity during cell death was supported by the observations that chemical inhibitors that reduce VAC also reduced the plasma membrane disruption induced by TNF-alpha in wild-type L929 cells, while increases in VAC due to genetic mutation, senescence, cell culture conditions, and chemical inhibitors all changed the morphology of cell death from one with an originally nearly intact plasma membrane to one with membrane disruption in a number of different cells. Moreover, the ATP depletion-mediated change from apoptosis to necrosis is also associated with the increases of VAC. The increase in lysosomal size may due to intracellular self-digestion of dying cells. Big lysosomes are easy to rupture, and the release of hydrolytic enzymes from ruptured lysosomes can cause plasma membrane disruption.

Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factor-alpha.

Macrophages from different inbred mouse strains exhibit striking differences in their sensitivity to anthrax lethal toxin (LeTx)-induced cytolysis. Although LeTx-induced cytolysis of macrophages plays an important role in the outcome of anthrax infection, the sensitivity of macrophages in vitro does not correlate with in vivo susceptibility to infection of Bacillus anthracis. This divergence suggests that additional factors other than LeTx are involved in the cytolysis of LeTx-resistant macrophages in vivo. We found that LeTx-resistant macrophages became sensitive to LeTx-induced cytolysis when these cells were activated by bacterial components. Tumor necrosis factor-alpha induced by bacterial components was a key factor that cooperated with LeTx in inducing LeTx-resistant macrophage death. Tumor necrosis factor-alpha/LeTx-induced death of LeTx-resistant macrophages was dependent on mTor (mammalian target of rapamycin), but independent of caspases. Our data indicate that host responses to anthrax infection contribute to cytolysis of LeTx- resistant macrophages.