Circulating IgG antibodies to periodontal bacteria and lung cancer risk in the CLUE cohorts.

BACKGROUND: Oral health is a key indicator of overall health, well-being, and quality of life. Several studies have provided new evidence about the role of oral diseases, specifically periodontitis, in generating risk for various forms of cancers, including lung, colorectal, and pancreatic cancers. METHODS: Incident lung cancer cases (n = 192) and matched controls (n = 192) were selected from participants of the CLUE I and CLUE II cohorts. Archived serum samples collected from participants in 1974 (in CLUE I) were analyzed using immunoblotting for immunoglobulin G (IgG) antibody levels to 13 bacteria of the periodontium. Associations between antibody levels and lung cancer were estimated using conditional logistic regression. RESULTS: Most of the periodontal bacterial antibodies measured were inversely associated with lung cancer risk; of these, 3 were statistically significant (Prevotellaintermedia, Actinomyces naeslundii, and Veillonella parvula). A statistically significant positive association was observed for one of the Porphyromonas gingivalis strains after adjusting for P. intermedia. The sum of the logarithm of antibodies against the 13 measured bacteria was inversely associated with risk of lung cancer when the analysis was restricted to a longer follow-up (31-44 years after blood collection, highest vs lowest quartile: odds ratio = 0.26, 95% confidence interval = 0.08 to 0.84). CONCLUSIONS: Findings from this study highlight the complexity of using serum IgG antibodies to periodontal bacteria to identify associations between oral pathogens and risk of lung cancer. The inverse associations observed for antibodies to periodontal bacteria suggest that these may represent markers of immunity that provide some advantage in reducing the development of lung cancer.

Antibodies to oral pathobionts and colon cancer risk in the CLUE I cohort study.

Periodontitis has been associated with an increased risk for gastrointestinal cancers. The objective of our study was to investigate the association of antibodies to oral bacteria and the risk of colon cancer in a cohort setting. Using the CLUE I cohort, a prospective cohort initiated in 1974 in Washington County, Maryland, we conducted a nested case-control study to examine the association of levels of IgG antibodies to 11 oral bacterial species (13 total strains) with risk of colon cancer diagnosed a median of 16 years later (range: 1-26 years). Antibody response was measured using checkerboard immunoblotting assays. We included 200 colon cancer cases and 200 controls matched on age, sex, cigarette smoking status, time of blood draw and pipe or cigar smoking status. Controls were selected using incidence density sampling. Conditional logistic regression models were used to assess the association between antibody levels and colon cancer risk. In the overall analysis, we observed significant inverse associations for 6 of the 13 antibodies measured (P-trends <.05) and one positive association for antibody levels to Aggregatibacter actinomycetemcomitans (ATCC 29523; P-trend = .04). While we cannot rule out a role for periodontal disease in colon cancer risk, findings from our study suggest that a strong adaptive immune response may be associated with a lower risk of colon cancer. More studies will need to examine whether the positive associations we observed with antibodies to A. actinomycetemcomitans reflect a true causal association for this bacterium.

Corneal fibrosis abrogation by a localized AAV-mediated inhibitor of differentiation 3 (Id3) gene therapy in rabbit eyes in vivo.

Previously we found that inhibitor of differentiation 3 (Id3) gene, a transcriptional repressor, efficiently inhibits corneal keratocyte differentiation to myofibroblasts in vitro. This study evaluated the potential of adeno-associated virus 5 (AAV5)-mediated Id3 gene therapy to treat corneal scarring using an established rabbit in vivo disease model. Corneal scarring/fibrosis in rabbit eyes was induced by alkali trauma, and 24 h thereafter corneas were administered with either balanced salt solution AAV5-naked vector, or AAV5-Id3 vector (n = 6/group) via an optimized reported method. Therapeutic effects of AAV5-Id3 gene therapy on corneal pathology and ocular health were evaluated with clinical, histological, and molecular techniques. Localized AAV5-Id3 gene therapy significantly inhibited corneal fibrosis/haze clinically from 2.7 to 0.7 on the Fantes scale in live animals (AAV5-naked versus AAV5-Id3; p < 0.001). Furthermore, AAV5-Id3 treatment significantly reduced profibrotic gene mRNA levels: α-smooth muscle actin (α-SMA) (2.8-fold; p < 0.001), fibronectin (3.2-fold; p < 0.001), collagen I (0.8-fold; p < 0.001), and collagen III (1.4-fold; p < 0.001), as well as protein levels of α-SMA (23.8%; p < 0.001) and collagens (1.8-fold; p < 0.001). The anti-fibrotic activity of AAV5-Id3 is attributed to reduced myofibroblast formation by disrupting the binding of E-box proteins to the promoter of α-SMA, a transforming growth factor-ß signaling downstream target gene. In conclusion, these results indicate that localized AAV5-Id3 delivery in stroma caused no clinically relevant ocular symptoms or corneal cellular toxicity in the rabbit eyes.

Exodontia associated bacteremia in horses characterized by next generation sequencing.

Bacteremia resulting from dental surgery is increasingly recognized as a health risk, especially in older and immunocompromised patients. Dentistry-associated bacteremia can lead to remote infections, as exemplified by valvular endocarditis. Emerging evidence points to a novel role played by oral cavity commensals in the pathogenesis of diabetes, respiratory disease, cardiovascular disease, and adverse pregnancy outcomes. Whether dental extraction, a commonly undertaken procedure in old horses, causes bacteremia has not been reported extensively. In a prospective clinical study using next generation sequencing (based on bacterial 16S rRNA), the circulating blood microbiome was characterized before and at 1 h following extraction of incisor, canine or cheek teeth from 29 adult horses with dental disease. 16S rRNA gene sequencing results from the blood microbiome were compared with those from gingival swab samples obtained prior to extraction at the location of the diseased tooth. Bacteremia associated with translocated gingival commensals was demonstrated in horses undergoing exodontia and was, in some cases, still evident one hour post-operatively.

Novel insights into gene therapy in the cornea.

Corneal disease remains a leading cause of impaired vision world-wide, and advancements in gene therapy continue to develop with promising success to prevent, treat and cure blindness. Ideally, gene therapy requires a vector and gene delivery method that targets treatment of specific cells or tissues and results in a safe and non-immunogenic response. The cornea is a model tissue for gene therapy due to its ease of clinician access and immune-privileged state. Improvements in the past 5-10 years have begun to revolutionize the approach to gene therapy in the cornea with a focus on adeno-associated virus and nanoparticle delivery of single and combination gene therapies. In addition, the potential applications of gene editing (zinc finger nucleases [ZNFs], transcription activator-like effector nucleases [TALENs], Clustered Regularly Interspaced Short Palindromic Repeats/Associated Systems [CRISPR/Cas9]) are rapidly expanding. This review focuses on recent developments in gene therapy for corneal diseases, including promising multiple gene therapy, while outlining a practical approach to the development of such therapies and potential impediments to successful delivery of genes to the cornea.

Role of inhibitor of differentiation 3 gene in cellular differentiation of human corneal stromal fibroblasts.

Purpose: Inhibitor of differentiation (Id) proteins are helix-loop-helix (HLH) transcriptional repressors that modulate a range of developmental and cellular processes, including cell differentiation and cell cycle mobilization. The inhibitor of differentiation 3 (Id3) gene, a member of the Id gene family, governs the expression and progression of transforming growth factor beta (TGFß)-mediated cell differentiation. In the face of mechanical, chemical, or surgical corneal insults, corneal keratocytes differentiate into myofibroblasts for wound repair. Excessive development or persistence or both of myofibroblasts after wound repair results in corneal haze that compromises corneal clarity and visual function. The objective of this study was to investigate whether Id3 overexpression in human corneal stromal fibroblasts governs TGFß-driven cellular differentiation and inhibits keratocyte to myofibroblast transformation. Methods: Primary human corneal stromal fibroblast (h-CSF) cultures were generated from donor human corneas. Human corneal myofibroblasts (h-CMFs) were produced by growing h-CSF in the presence of TGFß1 under serum-free conditions. The Id3 gene was cloned into a mammalian expression vector (pcDNA3 mCherry LIC cloning vector), and the nucleotide sequence of the vector constructs was confirmed with sequencing as well as through restriction enzyme analysis. The Id3 mammalian overexpression vector was introduced into h-CSFs using a lipofectamine transfection kit. The expression of Id3 in selected clones was characterized with quantitative real-time PCR (qRT-PCR), immunocytochemistry, and western blotting. Phase contrast microscopy and trypan blue exclusion assays were used to evaluate the effects of the transfer of the Id3 gene on the hCSF phenotype and viability, respectively. To analyze the inhibitory effects of the Id3 gene transfer on TGFß-induced formation of h-CMFs, expression of the mRNA and protein of the myofibroblast marker alpha smooth muscle actin (α-SMA) was examined with qRT-PCR, western blotting, and immunocytochemistry. Student t test, analysis of variance (ANOVA), and Bonferroni adjustment for repeated measures were used for statistical analysis. Results: The results indicate that Id3 overexpression does not alter the cellular phenotype or viability of h-CSFs. Overexpression of the Id3 gene in h-CSF cells grown in the presence of TGFß1 under serum-free conditions showed a statistically significant decrease (76.3±4.3%) in α-SMA expression (p<0.01) compared to the naked-vector transfected or non-transfected h-CSF cells. Id3-transfected, naked-vector transfected, and non-transfected h-CSF cells grown in the absence of TGFß1 showed the expected low expression of α-SMA (0-5%). Furthermore, Id3 overexpression statistically significantly decreased TGFß-induced mRNA levels of profibrogenic genes such as fibronectin, collagen type I, and collagen type IV (1.80±0.26-, 1.70±0.35- and 1.70±0.36-fold, respectively; p<0.05) that a play role in stromal matrix modulation and corneal wound healing. Results of the protein analysis with western blotting indicated that Id3 overexpression in h-CSF cells effectively slows TGFß-driven differentiation and formation of h-CMFs. Results for subsequent overexpression studies showed that this process occurs through the regulation of E2A, a TATA box protein. Conclusions: Id3 regulates TGFß-driven differentiation of h-CSFs and formation of h-CMFs in vitro. Targeted Id3 gene delivery has potential to treat corneal fibrosis and reestablish corneal clarity in vivo.

A Novel Topical Ophthalmic Formulation to Mitigate Acute Mustard Gas Keratopathy In Vivo: A Pilot Study.

Purpose: This pilot study investigated the in vivo therapeutic potential and tolerability of a multimodal ophthalmic formulation, topical eye drops (TED), for acute mustard gas keratopathy (MGK) using a rabbit model. Methods: Twenty New Zealand White rabbits were used. Only right eyes of 18 rabbits (oculus dexter [OD]) received single sulfur mustard gas (SM) vapor injury, whereas contralateral eyes were left untreated or received TED for tolerabilty evaluation. Two rabbit eyes received no treatment and served as age-matched naive control. The four groups were: Naive (oculus sinister [OS] untreated eyes; n = 9); TED (OS treated only with TED BID for 3 days; n = 9); SM (OD exposed to SM vapor; n = 9); and SM+TED (OD exposed to SM+TED BID for 3 days; n = 9). Ocular examination in live rabbits were performed utilizing slit-lamp biomicroscopy, Fantes grading system, fluorescein staining, Schirmer's tests, pachymetry, and applanation tonometry. Cellular and molecular changes in rabbit corneas were assessed after humane euthanasia on day-3 and day-7 with histopathological and real-time polymerase chain reaction PCR techniques. Results: TED to rabbit eyes was found tolerable in vivo. SM-exposed eyes showed significant increase in Fantes scores, central corneal thickness (CCT), Schirmer's test, epithelium-stroma separation, and corneal edema. TED mitigated clinical symptoms by reducing corneal edema, Fantes scores, CCT, and Schirmer's test. Further, TED decreased SM-induced corneal haze, inflammatory and profibrotic markers, transforming growth factor-TGF-ß1 and cyclooxygenase-2COX-2, and damage to corneal structure, including epithelial-stromal integrity. Conclusions: The developed multimodal eyedrop formulation, TED, has potential to mitigate acute MGK effectively in vivo. Translational Relevance: TED is effective against MGK.

Effects of Orally Administered Resveratrol on TNF, IL-1ß, Leukocyte Phagocytic Activity and Oxidative Burst Function in Horses: A Prospective, Randomized, Double-Blinded, Placebo-Controlled Study.

Resveratrol, a phytophenol, is a commonly used equine nutraceutical supplement touted to exert anti-inflammatory effects. The effect of orally administered resveratrol on tumor necrosis factor (TNF), interleukin-1ß (IL-1ß), leukocyte phagocytic activity or oxidative burst function have not been reported in horses. The objective of this study was to determine the effects of a commercially available, orally administered resveratrol product on innate immune functions in healthy adult horses. Whole blood was collected from 12 horses prior to and following 3 weeks of treatment with either the manufacturer's recommended dose of resveratrol or placebo. Phagocytosis, oxidative burst and pathogen associated molecular pattern (PAMP) motif-stimulated leukocyte production of TNF and IL-1ß were compared pre- and post-treatment between treatment groups. Phagocytosis and oxidative burst capacity were evaluated via flow cytometry. Tumor necrosis factor and IL-1ß were measured using cytotoxicity and ELISA assays, respectively. There were no significant differences in phagocytosis, oxidative burst or stimulated TNF or IL-1ß production between resveratrol and placebo treatment groups. Orally administered resveratrol at a routinely recommended dose for a duration of 3 weeks did not significantly affect phagocytic activity, oxidative burst function or PAMP-stimulated leukocyte cytokine production.

Investigation of the potential immunomodulatory effects of resveratrol on equine whole blood: An in vitro investigation.

Horses affected with gastrointestinal conditions such as colic or colitis are at substantial risk for translocation of bacterial components such as lipopolysaccharide (LPS, endotoxin) from the gastrointestinal tract into circulation resulting in systemic inflammation and subsequent morbidity and mortality. Therefore, there is a need for effective preventive and treatment strategies aimed at minimizing the host's inflammatory reaction to these pathogen-associated molecular patterns (PAMPs) from gastrointestinal disease. Resveratrol (RES, trans-3,5,4'-trihydroxystilbene) is a phytoalexin commonly found in fruits and beverages, including red wine. Health benefits associated with the consumption of red wine have been attributed to RES. Resveratrol has been significantly shown to exert a powerful anti-inflammatory effect in laboratory animals subjected to experimental endotoxemia/sepsis. Therefore, the objective of this study was to determine in vitro whether RES had an inhibitory effect on the production of tumor necrosis factor (TNF) in cultivated whole blood (Cwb) following stimulation by PAMPs. We hypothesized that RES would inhibit TNF production in Cwb following stimulation by LPS or lipoteichoic acid (LTA). Production of TNF bioactivity in Cwb was measured in the presence of phosphate buffered saline (control), ethanol (solvent control), dexamethasone (anti-inflammatory control), LPS, LTA, and three different concentrations of RES. Both LPS and LTA stimulated TNF production, and addition of dexamethasone was inhibitory to this effect. An anti-inflammatory effect for RES was not demonstrated under the current experimental conditions. Further studies are required to characterize the effect of RES on the equine innate immune system during systemic inflammation.

Exposure to low dose ionising radiation: Molecular and clinical consequences.

This review article provides a comprehensive overview of the experimental data detailing the incidence, mechanism and significance of low dose hyper-radiosensitivity (HRS). Important discoveries gained from past and present studies are mapped and highlighted to illustrate the pathway to our current understanding of HRS and the impact of HRS on the cellular response to radiation in mammalian cells. Particular attention is paid to the balance of evidence suggesting a role for DNA repair processes in the response, evidence suggesting a role for the cell cycle checkpoint processes, and evidence investigating the clinical implications/relevance of the effect.

Exposure to low dose ionising radiation: molecular and clinical consequences.

This review article provides a comprehensive overview of the experimental data detailing the incidence, mechanism and significance of low dose hyper-radiosensitivity (HRS). Important discoveries gained from past and present studies are mapped and highlighted to illustrate the pathway to our current understanding of HRS and the impact of HRS on the cellular response to radiation in mammalian cells. Particular attention is paid to the balance of evidence suggesting a role for DNA repair processes in the response, evidence suggesting a role for the cell cycle checkpoint processes, and evidence investigating the clinical implications/relevance of the effect.

DNA mismatch repair protein MSH2 dictates cellular survival in response to low dose radiation in endometrial carcinoma cells.

DNA repair and G2-phase cell cycle checkpoint responses are involved in the manifestation of hyper-radiosensitivity (HRS). The low-dose radioresponse of MSH2 isogenic endometrial carcinoma cell lines was examined. Defects in cell cycle checkpoint activation and the DNA damage response in irradiated cells (0.2 Gy) were evaluated. HRS was expressed solely in MSH2+ cells and was associated with efficient activation of the early G2-phase cell cycle checkpoint. Maintenance of the arrest was associated with persistent MRE11, γH2AX, RAD51 foci at 2 h after irradiation. Persistent MRE11 and RAD51 foci were also evident 24 h after 0.2 Gy. MSH2 significantly enhances cell radiosensitivity to low dose IR.

Exposure to hypoxia following irradiation increases radioresistance in prostate cancer cells.

BACKGROUND: Tumor hypoxia is a common feature of prostate tumors associated with the stabilization of hypoxia-inducible-factor 1alpha (HIF-1α) and poor prognosis following radiation therapy. Lack of oxygen at the time of irradiation is associated with radioresistance, but recent reports suggest radioresponse is also modulated by the dynamic nature of tumor hypoxia. OBJECTIVE: We proposed to evaluate the effect of post-irradiation hypoxic exposure on the radioresponse of 2 prostate cancer (CaP) cell lines (22Rv1, DU145) and to examine whether it correlates with modified cellular responses induced by hypoxia. METHODS AND RESULTS: Aerobic and hypoxic CaP cells exposed to hypoxia (24 h) after irradiation (4 Gy) gained a survival advantage compared with cells fully oxygenated after irradiation. This survival advantage was associated with induction of a G2/M cell cycle arrest, reduced induction of apoptosis and decreased amount of senescent cells. These modified cellular responses appeared mediated by HIF-1α. CONCLUSION: Our data suggest that targeting hypoxia after irradiation may benefit patients with aggressive hypoxic prostate tumors.

Docetaxel maintains its cytotoxic activity under hypoxic conditions in prostate cancer cells.

OBJECTIVE: The efficacy of docetaxel has recently been shown to be increased under hypoxic conditions through the down-regulation of hypoxia-inducible-factor 1α (HIF1A). Overexpression of the hypoxia-responsive gene class III ß-tubulin (TUBB3) has been associated with docetaxel resistance in a number of cancer models. We propose that administration of docetaxel to prostate patients has the potential to reduce the hypoxic response through HIF1A down-regulation and that TUBB3 down-regulation participates in sensitivity to docetaxel. METHODS: The cytotoxic effect of docetaxel was determined in both 22Rv1 and DU145 prostate cancer cell lines and correlated with HIF1A expression levels under aerobic and hypoxic conditions. Hypoxia-induced chemoresistance was investigated in a pair of isogenic docetaxel-resistant PC3 cell lines. Basal and hypoxia-induced TUBB3 gene expression levels were determined and correlated with methylation status at the HIF1A binding site. RESULTS: Prostate cancer cells were sensitive to docetaxel under both aerobic and hypoxic conditions. Hypoxic cytotoxicity of docetaxel was consistent with a reduction in detected HIF1A levels. Sensitivity correlated with reduced basal and hypoxia-induced HIF1A and TUBB3 expression levels. The TUBB3 HIF1A binding site was hypermethylated in prostate cell lines and tumor specimens, which may exclude transcription factor binding and induction of TUBB3 expression. However, acquired docetaxel resistance was not associated with TUBB3 overexpression. CONCLUSION: These data suggest that the hypoxic nature of a tumor may have relevance as regard to their response to docetaxel. Further investigation into the nature of this relationship may allow identification of novel targets to improve tumor control in prostate cancer patients.

Microtubule-targeting-compound PBOX-15 radiosensitizes cancer cells in vitro.

BACKGROUND: We proposed to investigate the radiosensitizing properties of PBOX-15, a novel microtubule-disrupting agent, in a panel of cancer cell lines. RESULTS: PBOX-15 treatment was associated with significant cell kill and increased radiosensitivity in all three cell lines tested. The number of surviving cells in response to the combined treatment was significantly less than PBOX -15 alone in 22Rv1 cells. In these cells, radiosensitisation correlated with induction of G2/M cell cycle arrest by PBOX-15. The compound sustained its activity and increased HIF-1Α expression under hypoxic conditions. PBOX-15 prevented onset of hypoxia-induced radioresistance in hypoxic prostate cells and reduced the surviving fraction of irradiated hypoxic cells to levels similar to those achieved under aerobic conditions. METHODS: Clonogenic assays were used to determine sensitivity of a panel of cancer cell lines (22Rv1, A549, U87) to PBOX-15 alone or in combination with a single 2Gy dose fraction. Induction of cell cycle arrest and apoptosis was investigated in 22Rv1 prostate cancer cells. The cytotoxic properties of the compound under hypoxic conditions were correlated with Hypoxia Inducible Factor 1 alpha (HIF-1Α) gene and protein expression levels and its radiosensitisation potential was investigated in hypoxic 22Rv1 using clonogenic assays. CONCLUSIONS: This preliminary data identifies the potential of PBOX-15 as a novel radiosensitising agent for the management of solid tumours and eradication of hypoxic cells.

DNA mismatch repair and the DNA damage response to ionizing radiation: making sense of apparently conflicting data.

The DNA mismatch repair (MMR) pathway detects and repairs DNA replication errors. While DNA MMR-proficiency is known to play a key role in the sensitivity to a number of DNA damaging agents, its role in the cytotoxicity of ionizing radiation (IR) is less well characterized. Available literature to date is conflicting regarding the influence of MMR status on radiosensitivity, and this has arisen as a subject of controversy in the field. The aim of this paper is to provide the first comprehensive overview of the experimental data linking MMR proteins and the DNA damage response to IR. A PubMed search was conducted using the key words "DNA mismatch repair" and "ionizing radiation". Relevant articles and their references were reviewed for their association between DNA MMR and IR. Recent data suggest that radiation dose and the type of DNA damage induced may dictate the involvement of the MMR system in the cellular response to IR. In particular, the literature supports a role for the MMR system in DNA damage recognition, cell cycle arrest, DNA repair and apoptosis. In this review we discuss our current understanding of the impact of MMR status on the cellular response to radiation in mammalian cells gained from past and present studies and attempt to provide an explanation for how MMR may determine the response to radiation.