Similarities and Differences between Osteoclast-Mediated Functional Activation of NK, CD3+ T, and γδ T Cells from Humans, Humanized-BLT Mice, and WT Mice.

This study is focused on assessing the activation in NK, CD3+ T, and γδ T cells when they interact with osteoclasts (OCs) and monocytes in the presence or absence of zoledronate (ZOL), both in humans and WT mice. OCs resulted in increased IFN-γ secretion in NK, CD3+ T, and γδ T cells, however, the significantly highest increase was seen when cells were co-cultured with ZOL-treated OCs. Our previous studies have demonstrated increased IFN-γ secretion in the peripheral blood-derived immune cells of bisphosphonate-related osteonecrosis of the jaw (BRONJ) mice model. This could be due to increased OCs-induced activation of immune cells with ZOL treatment. We also observed increased IFN-γ secretion in humanized-BLT (hu-BLT) mice NK cells when were co-cultured with OCs or monocytes, and higher IFN-γ secretion levels were seen in the presence of OCs or ZOL-treated OCs. In addition, similar effects on IFN-γ secretion levels of NK, CD3+ T, and γδ T cells were seen whether cells were co-cultured with allogeneic OCs or autologous OCs.

Supercharged NK Cell-Based Immuotherapy in Humanized Bone Marrow Liver and Thymus (Hu-BLT) Mice Model of Oral, Pancreatic, Glioblastoma, Hepatic, Melanoma and Ovarian Cancers.

In this paper, we review a number of in vitro and in vivo studies regarding the efficacy of supercharged NK (sNK) cell therapy in elimination or treatment of cancer. We have performed studies using six different types of cancer models of oral, pancreatic, glioblastoma, melanoma, hepatic and ovarian cancers using hu-BLT mice. Our in vitro studies demonstrated that primary NK cells preferentially target cancer stem-like cells (CSCs)/poorly differentiated tumors whereas sNK cells target both CSCs/poorly-differentiated and well-differentiated tumors significantly higher than primary activated NK cells. Our in vivo studies in humanized-BLT mice showed that sNK cells alone or in combination with other cancer therapeutics prevented tumor growth and metastasis. In addition, sNK cells were able to increase IFN-γ secretion and cytotoxic function by the immune cells in bone marrow, spleen, gingiva, pancreas and peripheral blood. Furthermore, sNK cells were able to increase the expansion and function of CD8+ T cells both in in vitro and in vivo studies. Overall, our studies demonstrated that sNK cells alone or in combination with other cancer therapeutics were not only effective against eliminating aggressive cancers, but were also able to increase the expansion and function of CD8+ T cells to further target cancer cells, providing a successful approach to eradicate and cure cancer.

Zoledronic acid mediated differential activation of NK cells in different organs of WT and Rag2-/- mice; stark differences between the bone marrow and gingivae.

We have previously shown that natural killer (NK) cells expand, and increase their function after interaction with cells that exhibit a number of different knock-down genes. We hypothesized that deletion or knockdown of a variety of key genes such as RAG may cause de-differentiation of the cells which could lead to increased NK expansion and function since we have shown previously that NK cells are activated and expanded by less differentiated cells. When comparing the function of NK cells from bone marrow (BM), spleen, pancreas, adipose tissue, and gingiva from WT mice to those from Rag2-/- mice, we observed a significant increase in IFN-γ secretion in all tissues of Rag2-/- mice versus in WT mice, with the exception of the gingivae in which similar levels were observed. After injecting WT mice with zoledronic acid (ZOL) and tooth extraction, immune cells from BM, spleen, and purified NK cells from spleen exhibited very high induction of IFN-γ and NK cell-mediated cytotoxicity with the exception of gingiva in which immune cells exhibited the opposite. In Rag2-/- mice, ZOL injection and tooth extraction stimulated IFN-γ secretion from BM immune cells but inhibited IFN-γ secretion from both spleen and gingivae. In both WT and Rag2-/- mice, immune cells from gingivae exhibited decreased IFN-γ secretion when activated, indicating significant regulation of immune cell function in the gingival microenvironment. However, even though significantly lower induction of IFN-γ was observed in both WT and Rag2-/- gingival cells after ZOL injection, ZOL mediated secretion of IFN-γ was still higher in the gingivae of WT mice when compared to those of Rag2-/- gingival cells. These results suggest an important role for IFN-γ in the pathogenesis of osteonecrosis lesions observed in post-tooth extraction jawbone.

Plasticity of Myeloid Cells during Oral Barrier Wound Healing and the Development of Bisphosphonate-related Osteonecrosis of the Jaw.

Injury to the barrier tissue initiates a rapid distribution of myeloid immune cells from bone marrow, which guide sound wound healing. Bisphosphonates, a widely used anti-bone resorptive drug with minimal systemic side effects, have been linked to an abnormal wound healing in the oral barrier tissue leading to, in some cases, osteonecrosis of the jaw (ONJ). Here we report that the development of ONJ may involve abnormal phenotypic plasticity of Ly6G+/Gr1+ myeloid cells in the oral barrier tissue undergoing tooth extraction wound healing. A bolus intravenous zoledronate (ZOL) injection to female C57Bl/6 mice followed by maxillary first molar extraction resulted in the development of ONJ-like lesion during the second week of wound healing. The multiplex assay of dissociated oral barrier cells exhibited the secretion of cytokines and chemokines, which was significantly modulated in ZOL mice. Tooth extraction-induced distribution of Ly6G+/Gr1+ cells in the oral barrier tissue increased in ZOL mice at week 2. ONJ-like lesion in ZOL mice contained Ly6G+/Gr1+ cells with abnormal size and morphology as well as different flow cytometric staining intensity. When anti-Ly6G (Gr1) antibody was intraperitoneally injected for 5 days during the second week of tooth extraction, CD11b+GR1(hi) cells in bone marrow and Ly6G+ cells in the oral barrier tissue were depleted, and the development of ONJ-like lesion was significantly attenuated. This study suggests that local modulation of myeloid cell plasticity in the oral barrier tissue may provide the basis for pathogenesis and thus therapeutic as well as preventive strategy of ONJ.

Osteonecrosis of the Jaw Developed in Mice: DISEASE VARIANTS REGULATED BY γδ T CELLS IN ORAL MUCOSAL BARRIER IMMUNITY.

Osteonecrosis of the jaw (ONJ), an uncommon co-morbidity in patients treated with bisphosphonates (BP), occurs in the segment of jawbone interfacing oral mucosa. This study aimed to investigate a role of oral mucosal barrier γδ T cells in the pathogenesis of ONJ. Female C57Bl/6J (B6) mice received a bolus zoledronate intravenous injection (ZOL, 540 µg/kg), and their maxillary left first molars were extracted 1 week later. ZOL-treated mice (WT ZOL) delayed oral wound healing with patent open wounds 4 weeks after tooth extraction with characteristic oral epithelial hyperplasia. γδ T cells appeared within the tooth extraction site and hyperplastic epithelium in WT ZOL mice. In ZOL-treated γδ T cell null (Tcrd(-/-) ZOL) mice, the tooth extraction open wound progressively closed; however, histological ONJ-like lesions were identified in 75 and 60% of WT ZOL and Tcrd(-/-) ZOL mice, respectively. Although the bone exposure phenotype of ONJ was predominantly observed in WT ZOL mice, Tcrd(-/-) ZOL mice developed the pustule/fistula disease phenotype. We further addressed the role of γδ T cells from human peripheral blood (h-γδ T cells). When co-cultured with ZOL-pretreated human osteoclasts in vitro, h-γδ T cells exhibited rapid expansion and robust IFN-γ secretion. When h-γδ T cells were injected into ZOL-treated immunodeficient (Rag2(-/-) ZOL) mice, the oral epithelial hyperplasia developed. However, Rag2(-/-) ZOL mice did not develop osteonecrosis. The results indicate that γδ T cells are unlikely to influence the core osteonecrosis mechanism; however, they may serve as a critical modifier contributing to the different oral mucosal disease variations of ONJ.

Augmentation of IFN-γ by bone marrow derived immune cells in the presence of severe suppression of IFN-γ in gingivae induced by zoledronic acid and denosumab in Hu-BLT mice model of ONJ.

Introduction: The potential mechanisms governing drug induced osteonecrosis of the jaw (ONJ) is not well understood, and is one of the objectives of this study. Thus, we tested the release of IFN-γ within different immune compartments including bone marrow and gingivae upon treatment with zoledronic acid (ZOL) and denosumab which are known to induce ONJ in susceptible individuals. Methods: We used humanized-BLT mouse model for the in-vivo studies reported in this paper. To determine the effects of zoledronic acid and denosumab on IFN-γ secretion and NK cell-mediated cytotoxicity; peripheral blood, bone marrow, spleen and gingiva were obtained after the injection of ZOL and denosumab in mice. Results: Percentages of B cells are much higher in wild-type mice whereas the proportions of immune subsets in humans and reconstituted hu-BLT peripheral-blood are similar. Therefore, hu-BLT mice are preferable model to study human disease, in particular, immune-pathologies induced by ZOL and denosumab. Both agents resulted in a severe suppression of IFN-γ in the gingiva, whereas they heightened the release of IFN-γ and NK cell-mediated cytotoxicity by the BM-derived immune cells. ZOL increased the IFN-γ secretion by the spleen and peripheral blood immune cells, whereas denosumab decreased the release IFN-γ by these cells significantly. Discussion: ZOL and denosumab may likely suppress IFN-γ secretion in gingiva through different mechanisms. In addition, to the suppression of IFN-γ secretion, denosumab mediated effect could in part be due to the decrease in the bone resorptive function of osteoclasts due to the induction of antibody dependent cellular cytotoxicity and lysis of osteoclasts, whereas ZOL is able to mediate cell death of osteoclasts directly. Suppression of IFN-gamma in gingiva is largely responsible for the inhibition of immune cell function, leading to dysregulated osteoblastic and osteoclastic activities. Restoration of IFN-gamma in the local microenvironment may result in establishment of homeostatic balance in the gingiva and prevention of osteonecrosis of jaw.

Potential rescue, survival and differentiation of cancer stem cells and primary non-transformed stem cells by monocyte-induced split anergy in natural killer cells.

Cytotoxic function of NK cells is largely suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. The aims of this review are to provide a rationale and a potential mechanism for immunosuppression in cancer and to demonstrate the significance of such immunosuppression in cellular differentiation and progression of cancer. We have recently shown that NK cells mediate significant cytotoxicity against primary oral squamous carcinoma stem cells (OSCSCs) as compared to their more differentiated oral squamous carcinoma cells. In addition, human embryonic stem cells, mesenchymal stem cells (hMSCs), dental pulp stem cells (hDPSCs) and induced pluripotent stem cells were all significantly more susceptible to NK-cell-mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB significantly augmented NK-cell function. Total population of monocytes and those depleted of CD16+ subsets were able to substantially suppress NK-cell-mediated lysis of OSCSCs, hMSCs and hDPSCs. Overall, our results suggest that stem cells but not their differentiated counterparts are significant targets of the NK-cell cytotoxicity. The concept of split anergy in NK cells and its contribution to cell differentiation, tissue repair and regeneration and in tumor resistance and progression will be discussed in this review.

Novel strategies to enhance survival and growth of pulp cells after dental restorations.

Using ex vivo human tooth, the authors demonstrated that dental pulp stromal cells that survive after placement of composite, mineral trioxide aggregate, and glass ionomer are weaker since they undergo synergistic cell death when exposed to 2-hydroxyethylmethacrylate. DPSCs extracted from teeth that were restored with the combination of composite or MTA or GI with N-acetyl cysteine were protectedfrom cell death. Therefore, application of NAC may protect the DPSCs from adverse effects after tooth restoration.

Phenotypic and Functional Alterations of Immune Effectors in Periodontitis; A Multifactorial and Complex Oral Disease.

Survival and function of immune subsets in the oral blood, peripheral blood and gingival tissues of patients with periodontal disease and healthy controls were assessed. NK and CD8 + T cells within the oral blood mononuclear cells (OBMCs) expressed significantly higher levels of CD69 in patients with periodontal disease compared to those from healthy controls. Similarly, TNF-α release was higher from oral blood of patients with periodontal disease when compared to healthy controls. Increased activation induced cell death of peripheral blood mononuclear cells (PBMCs) but not OBMCs from patients with periodontal disease was observed when compared to those from healthy individuals. Unlike those from healthy individuals, OBMC-derived supernatants from periodontitis patients exhibited decreased ability to induce secretion of IFN-γ by allogeneic healthy PBMCs treated with IL-2, while they triggered significant levels of TNF-α, IL-1ß and IL-6 by untreated PBMCs. Interaction of PBMCs, or NK cells with intact or NFκB knock down oral epithelial cells in the presence of a periodontal pathogen, F. nucleatum, significantly induced a number of pro-inflammatory cytokines including IFN-γ. These studies indicated that the relative numbers of immune subsets obtained from peripheral blood may not represent the composition of the immune cells in the oral environment, and that orally-derived immune effectors may differ in survival and function from those of peripheral blood.

N-acetylcysteine protects dental pulp stromal cells from HEMA-induced apoptosis by inducing differentiation of the cells.

Resin-based materials are now widely used in dental restorations. Although the use of these materials is aesthetically appealing to patients, it carries the risk of local and systemic adverse effects. The potential risks are direct damage to the cells and induction of immune-based hypersensitivity reactions. Dental pulp stromal cells (DPSCs) and oral keratinocytes are the major cell types which may come in contact with dental resins such as 2-hydroxyethyl methacrylate (HEMA) after dental restorations. Here we show that N-acetylcysteine (NAC) inhibits HEMA-induced apoptotic cell death and restores the function of DPSCs and oral epithelial cells. NAC inhibits HEMA-mediated toxicity through induction of differentiation in DPSCs, because the genes for dentin sialoprotein, osteopontin (OPN), osteocalcin, and alkaline phosphatase, which are induced during differentiation, are also induced by NAC. Unlike NAC, vitamins E and C, which are known antioxidant compounds, failed to prevent either HEMA-mediated cell death or the decrease in VEGF secretion by human DPSCs. More importantly, when added either alone or in combination with HEMA, vitamin E and vitamin C did not increase the gene expression for OPN, and in addition vitamin E inhibited the protective effect of NAC on DPSCs. NAC inhibited the HEMA-mediated decrease in NF-kappaB activity, thus providing a survival mechanism for the cells. Overall, the studies reported in this paper indicate that undifferentiated DPSCs have exquisite sensitivity to HEMA-induced cell death, and their differentiation in response to NAC resulted in an increased NF-kappaB activity, which might have provided the basis for their increased protection from HEMA-mediated functional loss and cell death.

Suppression of Gingival NK Cells in Precancerous and Cancerous Stages of Pancreatic Cancer in KC and BLT-Humanized Mice.

The aim of our studies is to determine the dynamics of natural killer (NK) cell modulation in gingivae in precancerous and cancerous stages of pancreatic and oral cancers in P48+/Cre;LSL-KRASG12D (KC) mice carrying a pancreas-specific oncogenic Kras mutation and BLT-humanized mice. Wild type and KC mice fed with control diet (CD) or high-fat calorie diet (HFCD), and the pancreatic and oral tumor-bearing humanized BLT (hu-BLT) mice were used to determine precancerous and cancer induced changes in numbers and function of gingival NK cells. Increased numbers of PanIN lesions and the greatest score of inflammation in pancreas of KC mice fed with CD and HFCD co-related with significant decline in percentages of circulating and gingival NK cells, lack of DX5+ NK expansion and increased secretion of IFN-γ and IL-6 after culture. At the malignant stage of pancreatic cancer, hu-BLT tumor-bearing mice had the lowest secretion of IFN-γ from cells dissociated from the gingival tissues as compared to those from non-tumor-bearing mice. Injection of NK cells into tumor-bearing mice increased IFN-γ secretion, and the secretion was similar or higher than those obtained by gingival cells from non-tumor-bearing hu-BLT control mice. The highest increase in IFN-γ secretion was observed when tumor-bearing mice were fed with AJ2 probiotic bacteria and injected with the NK cells. Along with an increase in secretion of IFN-γ, injection of NK cells in the presence and absence of feeding with AJ2 in pancreatic tumor-bearing mice increased percentages of CD45+ and CD3+ T cells in oral gingival cells. Similar results were observed with oral tumors. In conclusion, these results indicated that oral cavity may mirror systemic disease and provide a rationale for why cancer patients may be prone to suffer from diverse oral pathologies.

Disinfection Efficacy of Current Regenerative Endodontic Protocols in Simulated Necrotic Immature Permanent Teeth.

INTRODUCTION: The lack of mechanical debridement and reduced concentrations suggested for chemical debridement to maintain stem cell viability call into question the disinfection efficacy of current regenerative protocols. Current protocols vary in the concentration and type of antibiotic medicaments used. The aim of this study was to determine if simulated immature teeth infected with Enterococcus faecalis can be completely disinfected by following current standardized regenerative protocols and to evaluate the probable effects of residual bacteria on stem cell toxicity. METHODS: Sixty-eight caries-free maxillary incisors were used. S1 sampling protocols were validated in both negative and positive control groups via culture, scanning electron microscopy, and confocal laser scanning microscopy. All teeth, except the negative controls, were inoculated with E. faecalis. The teeth were divided into the following groups: group 1, triple antibiotic paste (ciprofloxacin:metronidazole:minocycline) at concentrations of 10, 1, and 0.1 mg/mL; group 2, double antibiotic paste (ciprofloxacin:metronidazole) at concentrations of 10, 1, and 0.1 mg/mL; group 3: Ultracal XS calcium hydroxide (Ultradent, St Louis, MO); and controls, negative and positive controls. Current regenerative protocols recommended by the American Association of Endodontists were followed. S2 sampling was performed after 4 weeks and tested for bacterial presence via culturing, scanning electron microscopy, and confocal laser scanning microscopic analysis. RESULTS: The data showed that calcium hydroxide and the current recommended antibiotic concentrations are not capable of completely eliminating bacteria from simulated necrotic immature permanent teeth. CONCLUSIONS: Overall, this study focuses on the need to re-evaluate the balance between stem cell toxicity and bacterial elimination in order to determine the appropriate concentrations and medicaments for successful regenerative endodontic procedures.

Split anergized Natural Killer cells halt inflammation by inducing stem cell differentiation, resistance to NK cell cytotoxicity and prevention of cytokine and chemokine secretion.

The mechanism of suppression of NK cytotoxicity in cancer patients is not clearly established. In this paper we provide evidence that anergized NK cells induce differentiation of healthy Dental Pulp Stem Cells (DPSCs) or transformed Oral Squamous Cancer Stem Cells (OSCSCs) resulting in cell growth inhibition, resistance to NK cell-mediated cytotoxicity and prevention of inflammatory mediators secretion. Induction of cytotoxicity resistance in differentiated cells correlated with increased CD54 and MHC class I surface expression and mediated by the combination of IFN-γ and TNF-α since antibodies to both, but not each cytokine alone, was able to inhibit resistance. In contrast, inhibition of cytokine and chemokine release was mediated by IFN-γ since the addition of anti-IFN-γ antibody, and not anti-TNF-α, restored secretion of inflammatory mediators in NK cell cultures with differentiated DPSCs and OSCSCs. There was a gradual and time dependent decrease in MHC class I and CD54 expression which correlated with the restoration of NK cell cytotoxicity, augmentation of cytokine secretion and increased cell growth from days 0-12 post NK removal. Continuous presence of NK cells is required for the maintenance of cell differentiation since the removal of NK cell-mediated function reverses the phenotype and function of differentiated cells to their stem-like cells.

Bisphosphonate-induced differential modulation of immune cell function in gingiva and bone marrow in vivo: role in osteoclast-mediated NK cell activation.

The aim of this study is to establish osteoclasts as key immune effectors capable of activating the function of Natural Killer (NK) cells, and expanding their numbers, and to determine in vivo and in vitro effect of bisphosphonates (BPs) during NK cell interaction with osteoclasts and on systemic and local immune function. The profiles of 27 cytokines, chemokines and growth factors released from osteoclasts were found to be different from dendritic cells and M1 macrophages but resembling to untreated monocytes and M2 macrophages. Nitrogen-containing BPs Zoledronate (ZOL) and Alendronate (ALN), but not non-nitrogen-containing BPs Etidronate (ETI), triggered increased release of pro-inflammatory mediators from osteoclasts while all three BPs decreased pit formation by osteoclasts. ZOL and ALN mediated significant release of IL-6, TNF-` and IL-1ß, whereas they inhibited IL-10 secretion by osteoclasts. Treatment of osteoclasts with ZOL inhibited NK cell mediated cytotoxicity whereas it induced significant secretion of cytokines and chemokines. NK cells lysed osteoclasts much more than their precursor cells monocytes, and this correlated with the decreased expression of MHC class I expression on osteoclasts. Intravenous injection of ZOL in mice induced pro-inflammatory microenvironment in bone marrow and demonstrated significant immune activation. By contrast, tooth extraction wound of gingival tissues exhibited profound immune suppressive microenvironment associated with dysregulated wound healing to the effect of ZOL which could potentially be responsible for the pathogenesis of Osteonecrosis of the Jaw (ONJ). Finally, based on the data obtained in this paper we demonstrate that osteoclasts can be used as targets for the expansion of NK cells with superior function for immunotherapy of cancer.

Induction of Split Anergy Conditions Natural Killer Cells to Promote Differentiation of Stem Cells through Cell-Cell Contact and Secreted Factors.

In this paper, we provide evidence that anergized NK cells through secreted factors and direct cell-cell contact have the ability to induce differentiation of healthy dental pulp stem cells and stem cell of apical papillae as well as transformed oral squamous cancer stem cell (OSCSC) and Mia-Paca-2, poorly differentiated stem-like pancreatic tumors, resulting in their resistance to NK cell-mediated cytotoxicity. Induction of NK cell resistance and differentiation in the stem cells correlated with the increased expression of CD54, B7H1, and MHC class I, and mediated by the combination of membrane-bound or secreted IFN-γ and TNF-α from the NK cells since antibodies to both cytokines and not each one alone were able to inhibit differentiation or resistance to NK cells. Similarly, antibodies to both TNF-α and IFN-γ were required to prevent NK-mediated inhibition of cell growth, and restored the numbers of the stem cells to the levels obtained when stem cells were cultured in the absence of anergized NK cells. Interestingly, the effect of anti-IFN-γ antibody in the absence of anti-TNF-α antibody was more dominant for the prevention of increase in surface receptor expression since its addition abrogated the increase in CD54, B7H1, and MHC class I surface expression. Antibodies to CD54 or LFA-1 was unable to inhibit differentiation whereas antibodies to MHC class I but not B7H1 increased cytotoxicity of well-differentiated oral squamous carcinoma cells as well as OSCSCs differentiated by the IL-2 + anti-CD16 mAb-treated NK cells whereas it inhibited the cytotoxicity of NK cells against OSCSCs. Thus, NK cells may inhibit the progression of cancer by killing and/or differentiation of cancer stem cells, which severely halt cancer growth, invasion, and metastasis.

Dual functions of natural killer cells in selection and differentiation of stem cells; role in regulation of inflammation and regeneration of tissues.

Accumulated evidence from our laboratory indicates that conditioned or anergized NK cells have the ability to induce resistance of healthy stem cells and transformed cancer stem cells through both secreted factors and direct cell-cell contact by inducing differentiation. Cytotoxic function of NK cells is suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. Furthermore, decreased peripheral blood NK cell function has been documented in many cancer patients. We have previously shown that NK cells mediate significant cytotoxicity against primary oral squamous carcinoma stem cells (OSCSCs) as compared to their more differentiated oral squamous carcinoma cells (OSCCs). In addition, human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSCs), human dental pulp stem cells (hDPSCs) and induced human pluripotent stem cells (hiPSCs) were all significantly more susceptible to NK cell mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or gene deletion of COX2 significantly augmented NK cell function. Furthermore, the induction of resistance of the stem cells to NK cell mediated cytotoxicity and their subsequent differentiation is amplified when either the stem cells or the NK cells were cultured in the presence of monocytes. Therefore, we propose that the two stages of NK cell maturation namely CD16+CD56dimCD69- NK cells are important for the lysis of stem cells or poorly differentiated cells whereas the CD16dim/-CD56dim/+CD69+NK cells are important for differentiation and eventual regeneration of the tissues and the resolution of inflammation, thus functionally serving as regulatory NK cells (NK(reg)). CD16 receptor on the NK cells were found to be the receptor with significant potential to induce NK cell anergy, however, our recent data indicated that NKp46 but not NKp30 or NKp44 were also able to induce significant anergy in NK cells, although the levels were less when compared to CD16 receptor triggering. The concept of split anergy in NK cells and generation of NK(reg) and its contribution to cell differentiation, tissue repair and regeneration and in tumor resistance will be discussed in this review.

The influence of glutathione on redox regulation by antioxidant proteins and apoptosis in macrophages exposed to 2-hydroxyethyl methacrylate (HEMA).

Resin monomers like 2-hydroxyethyl methacrylate (HEMA) disturb cell functions including responses of the innate immune system, mineralization and differentiation, or induce cell death via apoptosis. These phenomena are associated with oxidative stress and a reduction in the concentration of the antioxidant glutathione (GSH), resulting in imbalanced redox homeostasis. Thus far, the precise mechanism of how resin monomers interfere with cellular redox regulation is unknown. The present study provides insight into the induction of apoptosis and the differential expression of antioxidant enzymes depending on the availability of GSH. Buthionine sulfoximine (BSO) was used to inhibit GSH synthesis, while 2-oxothiazolidine-4-carboxylate (OTC), and N-acetylcysteine (NAC) as prodrugs supported GSH synthesis in RAW264.7 mouse macrophages exposed to HEMA (0-8 mm) for 24 h. The level of GSH was significantly decreased after cells were preincubated with BSO, and the formation of reactive oxygen species (ROS) increased in cultures subsequently exposed to HEMA. Apoptosis was drastically increased by BSO in HEMA-exposed cell cultures as well, but OTC and NAC retracted HEMA-induced cell death. These results show that dental monomer-induced apoptosis is causally related to the availability of GSH. The hydrogen peroxide decomposing enzymes glutathione peroxidase (GPx1/2) and catalase were differentially regulated in HEMA-exposed cultures. Expression of GPx1/2 was inhibited by HEMA and further reduced in the presence of BSO. SOD1 (superoxide dismutase) expression was inhibited in the presence of HEMA, and was decreased to an even greater extent by BSO, possibly due to H(2)O(2)-feedback inhibition. The expression of catalase was considerably up-regulated in HEMA-exposed cultures, implying that H(2)O(2) is the type of ROS that is significantly increased in monomer-exposed cells. OTC and NAC counteracted the effect of HEMA on GPx1/2, SOD1, and catalase expression. HO-1 (heme oxygenase) expression was strongly enhanced by HEMA, suggesting the need for further antioxidants like bilirubin to support enzyme activities that directly regulate H(2)O(2) equilibrium. Expression of the oxidoreductase thioredoxin (TRX1), the second major thiol-dependent antioxidant system in eukaryotic cells, was slightly reduced, while the oxygen-sensing protein HIF-1α was downregulated in HEMA-exposed cell cultures. These results indicate that cells and tissues actively respond to monomer-induced oxidative stress by the differential expression of enzymatic antioxidants.

Natural killer cells preferentially target cancer stem cells; role of monocytes in protection against NK cell mediated lysis of cancer stem cells.

Mounting effective anti-tumor immune responses by cytotoxic effectors is important for the clearance of tumors. However, accumulated evidence suggests that the cytotoxic function of immune effectors is largely suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. The aims of this review are to provide a rationale and potential mechanism for immunosuppression in cancer, and to demonstrate the significance of such immunosuppression in cellular differentiation and tissue regeneration in pathological conditions, and progression of cancer. We have recently shown that increased NK cell function was seen when they were cultured with primary oral squamous carcinoma stem cells (OSCSCs) as compared to their more differentiated oral squamous carcinoma cells (OSCCs). In addition, human embryonic stem cells (hESCs), Mesenchymal Stem Cells (hMSCs), dental pulp stem cells (hDPSCs) and induced pluripotent stem cells (hiPSCs) were significantly more susceptible to NK cell mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 augmented NK cell function significantly. Total population of monocytes and those depleted of CD16(+) subsets were able to substantially prevent NK cell mediated lysis of OSCSCs, MSCs and DPSCs. Taken together, our results suggest that stem cells are significant targets of the NK cell cytotoxicity. The concept of split anergy in NK cells and its contribution to tissue repair and regeneration and in tumor resistance and progression will be discussed in this review. Therefore, patients with cancer may benefit from repeated allogeneic NK cell transplantation at the site of the tumor for specific elimination of cancer stem cells.

Tumor induced inactivation of natural killer cell cytotoxic function; implication in growth, expansion and differentiation of cancer stem cells.

Accumulated evidence indicates that cytotoxic function of immune effectors is largely suppressed in the tumor microenvironment by a number of distinct effectors and their secreted factors. The aims of this review are to provide a rationale and a potential mechanism for immunosuppression in cancer and to demonstrate the significance of such immunosuppression in cellular differentiation and progression of cancer. To that end, we have recently shown that NK cells mediate significant cytotoxicity against primary oral squamous carcinoma stem cells (OSCSCs) as compared to their more differentiated oral squamous carcinoma cells (OSCCs). In addition, human embryonic stem cells (hESCs), Mesenchymal Stem Cells (hMSCs), dental pulp stem cells (hDPSCs) and induced pluripotent stem cells (hiPSCs) were all significantly more susceptible to NK cell mediated cytotoxicity than their differentiated counterparts or parental cells from which they were derived. We have also reported that inhibition of differentiation or reversion of cells to a less-differentiated phenotype by blocking NFκB or targeted knock down of COX2 in primary monocytes in vivo significantly augmented NK cell function. Total population of monocytes and those depleted of CD16(+) subsets were able to substantially prevent NK cell mediated lysis of OSCSCs, MSCs and DPSCs. Taken together, our results suggest that stem cells are significant targets of the NK cell cytotoxicity. The concept of split anergy in NK cells and its contribution to tissue repair and regeneration and in tumor resistance and progression will be discussed in this review.

The role of aggregation in Fusobacterium nucleatum- induced immune cell death.

INTRODUCTION: Fusobacterium nucleatum, an anaerobic oral bacterium, has been shown to be highly abundant in endodontic infections. Its role in these infections remains unclear. Previous studies have shown that F. nucleatum could aggregate immune cells. We have demonstrated that F. nucleatum can induce significant apoptosis in peripheral blood mononuclear cells (PBMCs). In this in vitro study, we sought to determine what role this aggregation phenomenon has on the induction of apoptosis in PBMCs. METHODS: F. nucleatum bacteria were treated as follows: formaldehyde-fixed, heat-treated, or sonicated before co-culturing with PBMCs. Cell aggregation and apoptosis of the PBMCs were assessed under light microscopy and analyzed by flow cytometry, respectively. PBMCs were then immobilized with a Matrigel matrix before treatment with F. nucleatum. Aggregation and apoptosis were assessed as before. Surface staining of activation marker CD69 was assessed by flow cytometry. The apoptosis and CD69 data underwent one-way analysis of variance, followed by post hoc Bonferroni test and χ(2) test, respectively, to determine statistical significance. RESULTS: Viable and formaldehyde-treated but not sonicated or heat-treated F. nucleatum bacteria were able to cause severe aggregation and apoptosis of the immune cells. Disruption of F. nucleatum mediated aggregation by immobilization of the cells with a Matrigel matrix resulted in a significant diminution of cell death but not cell activation when assessed by using surface expression of CD69 early activation antigen. CONCLUSIONS: F. nucleatum's ability to induce cell death in immune cells appears to be mediated through the immune cells being aggregated, which might have important implications for its pathogenesis.