Photobiomodulation of Gingival Cells Challenged with Viable Oral Microbes.

The oral cavity, a unique ecosystem harboring diverse microorganisms, maintains health through a balanced microflora. Disruption may lead to disease, emphasizing the protective role of gingival epithelial cells (GECs) in preventing harm from pathogenic oral microbes. Shifting GECs' response from proinflammatory to antimicrobial could be a novel strategy for periodontitis. Photobiomodulation therapy (PBMT), a nonpharmacologic host modulatory approach, is considered an alternative to drugs. While the host cell response induced by a single type of pathogen-associated molecular patterns (PAMPs) was widely studied, this model does not address the cellular response to intact microbes that exhibit multiple PAMPs that might modulate the response. Inspired by this, we developed an in vitro model that simulates direct interactions between host cells and intact pathogens and evaluated the effect of PBMT on the response of human gingival keratinocytes (HGKs) to challenge viable oral microbes at both the cellular and molecular levels. Our data demonstrated that LED pretreatment on microbially challenged HGKs with specific continuous wavelengths (red: 615 nm; near-infrared: 880 nm) induced the production of various antimicrobial peptides, enhanced cell viability and proliferation, promoted reactive oxygen species scavenging, and down-modulated proinflammatory activity. The data also suggest a potential explanation regarding the superior efficacy of near-infrared light treatment compared with red light in enhancing antimicrobial activity and reducing cellular inflammation of HGKs. Taken together, the findings suggest that PBMT enhances the overall barrier function of gingival epithelium while minimizing inflammation-mediated breakdown of the underlying structures.

Overexpression of secretory leukocyte peptidase inhibitor (SLPI) does not modulate experimental osteoarthritis but may be a biomarker for the disease.

OBJECTIVE: Osteoarthritic cartilage destruction can be regulated by the balance between proteases and anti-proteases. Here, we sought to identify novel cellular protease inhibitors associated with osteoarthritis (OA) pathogenesis. METHODS: Candidate molecules were screened from microarray data of chondrocytes treated with OA-associated catabolic factors. The functions of candidate molecules in OA pathogenesis were examined in primary-culture mouse articular chondrocytes and mouse models of OA, such as those stimulated by destabilization of the medial meniscus (DMM) or intra-articular (IA) injection of adenovirus expressing the candidate gene. The value of the selected candidate molecule as a biomarker of OA was examined by measuring its circulating levels in human and mouse blood. RESULTS: Bioinformatic analysis identified secretory leukocyte peptidase inhibitor (SLPI) as a highly upregulated cellular protease inhibitor in chondrocytes treated with pathogenic catabolic factors, including interleukin (IL)-1ß, hypoxia-inducible factor (HIF)-2α, and zinc importer ZIP8. The adenovirus-mediated overexpression of SLPI in joint tissues did not cause any OA-like change or modulate DMM- or HIF-2α-induced experimental OA in mice. SLPI also did not markedly modulate the expression of OA-associated catabolic or anabolic factors in chondrocytes. However, SLPI was specifically upregulated in OA cartilage, and the serum SLPI levels were significantly elevated in human OA patients and experimental OA mice, suggesting that SLPI may be a biomarker of OA. CONCLUSION: Although SLPI is upregulated in OA chondrocytes, it does not appear to per se modulate OA development in mice. However, it may be a potential biomarker of OA in humans and animal models.

Upregulation of Atrogin-1/FBXO32 is not necessary for cartilage destruction in mouse models of osteoarthritis.

OBJECTIVE: In a preliminary study, we found that recently identified catabolic regulators of osteoarthritis (OA), including hypoxia-inducible factor (HIF)-2α and members of the zinc-ZIP8-MTF1 axis, upregulate the E3 ubiquitin ligase, Atrogin-1 (encoded by Fbxo32), in chondrocytes. As the ubiquitination/proteasomal degradation pathways are tightly regulated to modulate the expression of catabolic factors in chondrocytes, we examined the in vivo functions of Atrogin-1 in mouse models of OA. METHODS: The mRNA and protein levels of Atrogin-1 and other regulators of OA were determined in primary cultured mouse chondrocytes, OA human cartilage, and OA cartilage from wild-type (WT) and Fbxo32-knockout (KO) mice subjected to destabilization of the medial meniscus or intra-articular (IA) injection of adenoviruses expressing HIF-2α (Ad-Epas1), ZIP8 (Ad-Zip8), or Atrogin-1 (Ad-Fbxo32). The effect of Atrogin-1 overexpression on the cartilage of WT mice was examined by IA injection of Ad-Fbxo32. RESULTS: Atrogin-1 mRNA levels in chondrocytes were markedly increased by treatment with interleukin-1ß, HIF-2α, and members of the zinc-ZIP8-MTF1 axis. Atrogin-1 protein levels were also increased in OA cartilage from humans and various mouse OA models. However, the forced overexpression of Atrogin-1 in chondrocytes did not modulate the expression of cartilage matrix molecules or matrix-degrading enzymes. Moreover, overexpression of Atrogin-1 in the mouse joint tissues failed to cause OA pathogenesis, and Fbxo32 knockout failed to affect post-traumatic OA cartilage destruction in mice. CONCLUSIONS: Although Atrogin-1 is upregulated in OA cartilage, overexpression of Atrogin-1 in the joint tissues or knockout of Fbxo32 does not affect OA cartilage destruction in mice.

Reciprocal regulation by hypoxia-inducible factor-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in osteoarthritis.

OBJECTIVE: Hypoxia-inducible factor-2α (HIF-2α) transcriptionally upregulates Nampt in articular chondrocytes. NAMPT, which exhibits nicotinamide phosphoribosyltransferase activity, in turn causes osteoarthritis (OA) in mice by stimulating the expression of matrix-degrading enzymes. Here, we sought to elucidate whether HIF-2α activates the NAMPT-NAD(+)-SIRT axis in chondrocytes and thereby contributes to the pathogenesis of OA. METHODS: Assays of NAD levels, SIRT activity, reporter gene activity, mRNA, and protein levels were conducted in primary cultured mouse articular chondrocytes. Experimental OA in mice was induced by intra-articular (IA) injection of adenovirus expressing HIF-2α (Ad-Epas1) or NAMPT (Ad-Nampt). The functions of SIRT in OA were examined by IA co-injection of SIRT inhibitors or adenovirus expressing individual SIRT isoforms or shRNA targeting specific SIRT isoforms. RESULTS: HIF-2α activated the NAMPT-NAD(+)-SIRT axis in chondrocytes by upregulating NAMPT, which stimulated NAD(+) synthesis and thereby activated SIRT family members. The activated NAMPT-SIRT pathway, in turn, promoted HIF-2α protein stability by negatively regulating its hydroxylation and 26S proteasome-mediated degradation, resulting in increased HIF-2α transcriptional activity. Among SIRT family members (SIRT1-7), SIRT2 and SIRT4 were positively associated with HIF-2α stability and transcriptional activity in chondrocytes. This reciprocal regulation was required for the expression of catabolic matrix metalloproteinases (MMP3, MMP12, and MMP13) and OA cartilage destruction caused by IA injection of Ad-Epas1 Ad-Nampt. CONCLUSION: The reciprocal regulation of HIF-2α and the NAMPT-NAD(+)-SIRT axis in articular chondrocytes is involved in OA cartilage destruction caused by HIF-2α or NAMPT.

In vitro differentiation of human liver-derived stem cells with mesenchymal characteristics into immature hepatocyte-like cells.

Liver transplantation is severely limited by donor shortage although it is the only effective treatment for end-stage liver disease. So the best alternative is hepatocyte transplantation. For obtaining human hepatocytes, some stem cells originating from extrahepatic or intraheptic tissues have been isolated and characterized. Previously we have reported that human liver-derived stem cells (HLSCs) could be isolated and expanded from donated livers unsuitable for transplantation; they expressed some markers of mesenchymal stem cells but neither hematopoietic nor oval cells. In this study, we isolated and expanded HLSCs with mesenchymal characteristics from another adult human liver. They showed mesenchymal morphology and grew well under serum condition similar to our previous reports. Also, they expressed some markers of mesenchymal stem cells, such as CD44, CD73, CD90, and CD105, through fluorescence-activated cell sorting analysis. When HLSCs were sequentially exposed to fibroblast growth factor-1 (FGF-1), FGF-4, and hepatocyte growth factor (HGF) followed by FGF-4, HGF, oncostatin M, and dexamethasone, they became round or polygonal, and expressed some hepatic markers such as albumin and α1-antitrypsin in the gene or protein level. Also, they showed urea synthesis activity 7 days after treatment of FGF-4, HGF, oncostatin M, and dexamethasone. These results provided that HLSCs would be a useful cell source in the field of regenerative medicine as well as liver cell biology.

RTOG 0518: randomized phase III trial to evaluate zoledronic acid for prevention of osteoporosis and associated fractures in prostate cancer patients.

BACKGROUND: RTOG 0518 evaluated the potential benefit of zoledronic acid therapy in preventing bone fractures for patients with high grade and/or locally advanced, non-metastatic prostate adenocarcinoma receiving luteinizing hormone-releasing hormone (LHRH) agonist and radiotherapy (RT). METHODS: Eligible patients with T-scores of the hip (<-1.0, but >-2.5 vs >-1.0) and negative bone scans were prospectively randomized to either zoledronic acid, 4 mg, concurrently with the start of RT and then every six months for a total of 6 infusions (Arm 1) or observation (Arm 2). Vitamin D and calcium supplements were given to all patients. Secondary objectives included quality of life (QOL) and bone mineral density (BMD) changes over a period of three years. RESULTS: Of 109 patients accrued before early closure, 96 were eligible. Median follow-up was 36.3 months for Arm 1 and 34.8 months for Arm 2. Only two patients experienced a bone fracture (one in each arm) resulting in no difference in freedom from any bone fracture (P=0.95), nor in QOL. BMD percent changes from baseline to 36 months were statistically improved with the use of zoledronic acid compared to observation for the lumbar spine (6% vs -5%, P<0.0001), left total hip (1% vs -8%, P=0.0002), and left femoral neck (3% vs -8%, P=0.0007). CONCLUSIONS: For patients with advanced, non-metastatic prostate cancer receiving LHRH agonist and RT, the use of zoledronic acid was associated with statistically improved BMD percent changes. The small number of accrued patients resulted in decreased statistical power to detect any differences in the incidence of bone fractures or QOL.

In vitro evaluation of migratory capacity of human liver stem cells influenced by soluble factors.

Although several studies have addressed the engraftment of stem cells into the liver, the exact mechanisms in vivo remain unclear. In this study, we investigated the effects of soluble factors on cell migration using purified, expanded human liver stem cells (HLSCs) obtained from a pediatric liver resection. Using a in vitro transwell migration assay, we evaluated the migratory capacity of HLSCs under the influence of the cytokines tumor necross factor- [TNF]-α, interleukin [IL]-6, and interferon (IFN)-γ or the growth factors vascular endothelial growth factor [VEGF], basic fibroblast growth factor [bFGF], and hepatocyte growth factor [HGF], which are known to be highly secreted during liver injury. We also evaluated the migratory capacity indirectly influenced by cryopreserved human hepatocytes. The migration across the transwell membrane was promoted by VEGF, bFGF, TNF-α, IFN-γ, or hepatocytes. The cryopreserved human hepatocytes especially induced significant migration. These results suggested the presence of unidentified soluble factors from hepatocytes. This experiment described a reliable system for quantitative migration studies to broaden our understanding of the directional nature of cell migration.

MPP(+) downregulates mitochondrially encoded gene transcripts and their activities in dopaminergic neuronal cells: protective role of Bcl-2.

The effects of neurotoxins on levels of mitochondrially encoded gene transcripts in a dopaminergic neuronal cell line, MN9D, were examined following treatment with 200 microM N-methyl-4-phenylpyridinium (MPP(+)) or 6-hydroxydopamine (6-OHDA). As confirmed by a Northern blot analysis, levels of cytochrome c oxidase subunit 3 (COX III) and ATPase subunit 6 (ATPase 6) transcript were decreased in a time-dependent manner following treatment with MPP(+) but not with 6-OHDA. Accordingly, enzymatic activity of cytochrome c oxidase (COX) and the intracellular ATP content were also decreased in MPP(+)-treated cells while these remained unaltered in 6-OHDA-treated cells. In the cell death paradigm induced by MPP(+), overexpression of Bcl-2 in MN9D cells (MN9D/Bcl-2) significantly blocked MPP(+)-induced downregulation of COX III and ATPase 6 transcripts. In MN9D/Bcl-2 cells, MPP(+)-induced downregulation of COX activity and the intracellular level of ATP was also blocked. Treatment with a pan-caspase inhibitor, however, neither prevented MPP(+)-induced downregulation of COX activity nor affected intracellular level of ATP in MN9D cells. Taken together, our present data suggest that Bcl-2 may play a regulatory role in energy metabolism by preventing downregulation of mitochondrially encoded gene(s) at a point distinct from its known anticaspase activity in MPP(+)-induced dopaminergic neuronal death.

Neutron radiation enhances cisplatin cytotoxicity independently of apoptosis in human head and neck carcinoma cells.

Recent advances in combined modality treatment of locally advanced head and neck cancer have improved local and regional disease control and survival with better functional outcome. However, the local and regional failure rate after radiation therapy is still high for tumors that respond poorly to cisplatin-based neoadjuvant chemotherapy. This clinical observation suggests a common biological mechanism for resistance to cisplatin and photon irradiation. In this report, we investigated the molecular basis underlying cisplatin resistance in head and neck squamous carcinoma (HNSCC) cells and asked if fast neutron radiation enhances cisplatin cytotoxicity in cisplatin-resistant cells. We found that cisplatin sensitivity correlates with caspase induction, a cysteine proteinase family known to initiate the apoptotic cell death pathway, suggesting that apoptosis may be a critical determinant for cisplatin cytotoxicity. Neutron radiation effectively enhanced cisplatin cytotoxicity in HNSCCs including cisplatin-resistant cells, whereas photon radiation had little effect on cisplatin cytotoxicity. Interestingly, neutron-enhanced cisplatin cytotoxicity was associated neither with apoptosis nor with cell cycle regulation, as determined by caspase activity assay, annexin V staining, and flow cytometric analysis. Taken together, the present study provides a molecular insight into cisplatin resistance and may also provide a basis for more effective multimodality protocols involving neutron radiation for patients with locally advanced head and neck cancer.

H-ras, but not N-ras, induces an invasive phenotype in human breast epithelial cells: a role for MMP-2 in the H-ras-induced invasive phenotype.

Elevated p21ras expression is associated with tumor aggressiveness in breast cancer including the extent of invasion into fat tissues, infiltration into lymphatic vessels and tumor recurrence. In the present study, we have examined the roles of H-ras and N-ras, members of the human ras gene family, in the pathogenesis of breast cancer. We show that H-ras, but not N-ras, induces an invasive phenotype in human breast epithelial cells (MCF10A) as determined by the Matrigel invasion assay, whereas both H-ras and N-ras induce anchorage-independent growth, as shown by soft agar assay. We examined the effects of H-ras and N-ras activation on the expression of MMP-2 and MMP-9, which can degrade type IV collagen, the major structural collagen of the basement membrane. We show that MMP-2 is efficiently induced by H-ras, whereas MMP-9 induction is more prominent in N-ras-activated MCF10A cells. We also show that H-ras-mediated invasiveness is significantly inhibited when the expression of MMP-2 is down-regulated, using an oligodeoxyribonucleotide complementary to the MMP-2 mRNA, or when MMP-2 activity is blocked by its inhibitor TIMP-2 (tissue inhibitors of matrix metalloproteinase-2). Our results show that the H-ras-induced invasive phenotype is associated more closely with the expression of MMP-2 in human breast epithelial cells, rather than the induction of MMP-9 expression, as shown previously for rat embryonic fibroblasts.

Ginsenoside RH-2 induces apoptotic cell death in rat C6 glioma via a reactive oxygen- and caspase-dependent but Bcl-X(L)-independent pathway.

We used the rat C6 gliomal cell line to investigate the potential role of ginsenoside Rh2 (G-Rh2) in brain tumor. G-Rh2 induced many apoptotic manifestations in C6 gliomal cells as evidenced by changes in cell morphology, generation of DNA fragmentation, activation of caspase and production of reactive oxygen species (ROS). As a result, cotreatment with antioxidants or a broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone effectively attenuated G-Rh2-induced cell death. However, specific cleavage of poly(ADP-ribose)polymerase into 85 kDa protein was not detected as demonstrated in many other apoptotic paradigms. Expression levels of Bcl-2 and Bax remained unchanged following G-Rh2 treatment. Furthermore, G-Rh2-induced cell death in C6 gliomal cells overexpressing antiapoptotic protein, Bcl-X(L), was comparable to that in parental cells. Taken together, our data indicate that G-Rh2-induced cell death is mediated by the generated ROS and the activation of caspase pathway in a Bcl-X(L)-independent manner.

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function.

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a "competence factor" to induce a set of early response genes expressed in G1 including p21WAF1/CIP1, a functional mediator of the tumor suppressor gene p53 in G1/S checkpoint. For PDGF-stimulated cells to progress beyond G1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G1/S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28v-sis expression vector, which was previously shown to activate PDGF alpha- and beta- receptors. Although the basal level of p21WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of "competence" growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo.

Effects of endoscopic variceal ligation in lower esophageal motor function: a prospective study.

OBJECTIVES: Endoscopic variceal ligation (EVL), a recently developed method for controlling active variceal bleeding and eradicating esophageal varices, has similar efficacy to endoscopic injection sclerotherapy (EIS) and is known to have a minimal risk of complications and fewer complications in the lower esophagus. However, since the site of EVL is chiefly done in the lower esophagus, we prospectively evaluated to investigate the effect of EVL on the lower esophageal motor function. METHODS: We evaluated the severity of esophageal varix with the endoscopy and the lower esophageal manometry in 27 patients who had no history of interventional therapy, for varices before EVL, 3 weeks and 6 months after the last EVL session. RESULTS: The EVL caused considerable diminution in the size of esophageal varix by a mean 8.2 (range 3-21) ligations in mean 1.7 (range 1-3) sessions. In most of the cases, the varices reappeared and enlarged when the procedure of EVL was stopped. There were two different types of changes (intermediate and late) in the lower esophageal motility. The intermediate post-EVL effects were the increase of peristaltic contraction amplitude and duration in the lower esophageal body after EVL. The late post-EVL effects were the prolongation of lower esophageal sphinctor (LES) relaxation duration and speedier peristaltic velocity in the lower esophageal body. CONCLUSIONS: We conclude from these findings that the intermediate post-EVL effect may be transient and the increase of peristaltic wave was due to diminution of esophageal varix.