Multiple Myeloma Derived Extracellular Vesicle Uptake by Monocyte Cells Stimulates IL-6 and MMP-9 Secretion and Promotes Cancer Cell Migration and Proliferation.

Multiple Myeloma (MM) is an incurable haematological malignancy caused by uncontrolled growth of plasma cells. MM pathogenesis is attributed to crosstalk between plasma cells and the bone marrow microenvironment, where extracellular vesicles (EVs) play a role. In this study, EVs secreted from a panel of MM cell lines were isolated from conditioned media by ultracentrifugation and fluorescently stained EVs were co-cultured with THP-1 monocyte cells. MM EVs from three cell lines displayed a differential yet dose-dependent uptake by THP-1 cells, with H929 EVs displaying the greatest EV uptake compared to MM.1s and U266 EVs suggesting that uptake efficiency is dependent on the cell line of origin. Furthermore, MM EVs increased the secretion of MMP-9 and IL-6 from monocytes, with H929 EVs inducing the greatest effect, consistent with the greatest uptake efficiency. Moreover, monocyte-conditioned media collected following H929 EV uptake significantly increased the migration and proliferation of MM cells. Finally, EV proteome analysis revealed differential cargo enrichment that correlates with disease progression including a significant enrichment of spliceosome-related proteins in H929 EVs compared to the U266 and MM.1s EVs. Overall, this study demonstrates that MM-derived EVs modulate monocyte function to promote tumour growth and metastasis and reveals possible molecular mechanisms involved.

Isolation and Characterization of Human Conjunctival Mesenchymal Stromal Cells and Their Extracellular Vesicles.

Purpose: The purpose of this study was to isolate and culture human conjunctival mesenchymal stromal cells (Conj-MSCs) from cadaveric donor tissue, and to obtain and characterize their extracellular vesicles (EVs) and their effect on conjunctival epithelium. Methods: Stromal cells isolated from cadaveric donor conjunctival tissues were cultured and analyzed to determine whether they could be defined as MSCs. Expression of MSC markers was analyzed by flow cytometry. Cells were cultured in adipogenic, osteogenic, and chondrocyte differentiation media, and stained with Oil Red, Von Kossa, and Toluidine Blue, respectively, to determine multipotent capacity. EVs were isolated from cultured Conj-MSCs by differential ultracentrifugation. EV morphology was evaluated by atomic force microscopy, size distribution analyzed by dynamic light scattering, and EVs were individually characterized by nanoflow cytometry. The effect of EVs on oxidative stress and viability was analyzed in in vitro models using the conjunctival epithelial cell line IM-HConEpiC. Results: Cultured stromal cells fulfilled the criteria of MSCs: adherence to plastic; expression of CD90 (99.95 ± 0.03% positive cells), CD105 (99.04 ± 1.43%), CD73 (99.99 ± 0.19%), CD44 (99.93 ± 0.05%), and absence of CD34, CD11b, CD19, CD45 and HLA-DR (0.82 ± 0.91%); and in vitro differentiation into different lineages. Main Conj-MSC EV subpopulations were round, small EVs that expressed CD9, CD63, CD81, and CD147. Conj-MSC EVs significantly decreased the production of reactive oxygen species in IM-HConEpiCs exposed to H2O2 in similar levels than adipose tissue-MSC-derived EVs and ascorbic acid, used as controls. Conclusions: It is possible to isolate human Conj-MSCs from cadaveric tissue, and to use these cells as a source of small EVs with antioxidant activity on conjunctival epithelial cells.

Cyclophilin A Isomerisation of Septin 2 Mediates Abscission during Cytokinesis.

The isomerase activity of Cyclophilin A is important for midbody abscission during cell division, however, to date, midbody substrates remain unknown. In this study, we report that the GTP-binding protein Septin 2 interacts with Cyclophilin A. We highlight a dynamic series of Septin 2 phenotypes at the midbody, previously undescribed in human cells. Furthermore, Cyclophilin A depletion or loss of isomerase activity is sufficient to induce phenotypic Septin 2 defects at the midbody. Structural and molecular analysis reveals that Septin 2 proline 259 is important for interaction with Cyclophilin A. Moreover, an isomerisation-deficient EGFP-Septin 2 proline 259 mutant displays defective midbody localisation and undergoes impaired abscission, which is consistent with data from cells with loss of Cyclophilin A expression or activity. Collectively, these data reveal Septin 2 as a novel interacting partner and isomerase substrate of Cyclophilin A at the midbody that is required for abscission during cytokinesis in cancer cells.

Phosphorylation of the prolyl isomerase Cyclophilin A regulates its localisation and release from the centrosome during mitosis.

The centrosome acts as a protein platform from which proteins are deployed to function throughout the cell cycle. Previously, we have shown that the prolyl isomerase Cyclophilin A (CypA) localizes to the centrosome in interphase and re-localizes to the midbody during mitosis where it functions in cytokinesis. In this study, investigation of CypA by SDS-PAGE during the cell cycle reveals that it undergoes a mobility shift during mitosis, indicative of a post-translational modification, which may correlate with its subcellular re-localization. Due to the lack of a phospho-specific antibody, we used site-directed mutagenesis to demonstrate that the previously identified serine 77 phosphorylation site within CypA is important for control of CypA centrosome localization. Furthermore, CypA is shown to interact with the mitotic NIMA-related kinase 2 (Nek2) during interphase and mitosis, while also interacting with the Nek2-antagonist PP1 during interphase but not during mitosis, suggesting a potential role for the Nek2-PP1 complex in CypA phospho-regulation. In support of this, Nek2 is capable of phosphorylating CypA in vitro. Overall, this work reveals that phosphorylation of CypA at serine 77 is important for its release from the centrosome during mitosis and may be regulated by the activity of Nek2 and PP1 during the cell cycle.

Extracellular Vesicles Isolated from Plasma of Multiple Myeloma Patients Treated with Daratumumab Express CD38, PD-L1, and the Complement Inhibitory Proteins CD55 and CD59.

Daratumumab (DARA) has improved the outcome of treatment of multiple myeloma (MM). DARA acts via complement-dependent and -independent mechanisms. Resistance to DARA may result from upregulation of the complement inhibitory proteins CD55 and CD59, downregulation of the DARA target CD38 on myeloma cells or altered expression of the checkpoint inhibitor ligand programmed death ligand-1 (PD-L1) or other mechanisms. In this study, EVs were isolated from peripheral blood (PB) and bone marrow (BM) from multiple myeloma (MM) patients treated with DARA and PB of healthy controls. EV size and number and the expression of CD38, CD55, CD59 and PD-L1 as well as the EV markers CD9, CD63, CD81, CD147 were determined by flow cytometry. Results reveal that all patient EV samples express CD38, PD-L1, CD55 and CD59. The level of CD55 and CD59 are elevated on MM PB EVs compared with healthy controls, and the level of PD-L1 on MM PB EVs is higher in patients responding to treatment with DARA. CD147, a marker of various aspects of malignant behaviour of cancer cells and a potential target for therapy, was significantly elevated on MM EVs compared with healthy controls. Furthermore, mass spectrometry data suggests that MM PB EVs bind DARA. This study reveals a MM PB and BM EV protein signature that may have diagnostic and prognostic value.

Investigation of canine extracellular vesicles in diffuse large B-cell lymphomas.

Diffuse large B-cell lymphomas (DLBCLs) are the most common lymphoproliferative diseases in dogs. DLBCL diagnosis to date has relied on histopathological analysis; however liquid biopsies have gained attention in recent years as a source of diagnostic and prognostic information. Liquid biopsies can be a source of circulating DNA, miRNA, circulating tumour cells or extracellular vesicles (EVs). In this study EVs were isolated from the plasma of healthy dogs, and dogs with lymphoma, and adenocarcinoma by iodixanol density gradient centrifugation. These EVs were positive for the EV markers CD63 and TSG101 and the pan-B cell markers CD79a, CD21, CD45, CD20. NTA analysis revealed that the DLBCL and adenocarcinoma dogs had elevated plasma EVs relative to the healthy dogs. Furthermore, the modal size of lymphoma EVs had decreased relative to healthy dogs while adenocarcinoma EVs were unchanged. This study demonstrates that the plasma EV population is altered in canine lymphoma patients in a manner similar to previous studies on human lymphomas. The similar changes to the EV population in dogs, together with the similar pathological features and treatment protocols in canine and human non-Hodgkin lymphomas would make dogs a good comparative model for studying the role of EVs in DLBCL development and progression.

Functional Antagonism of Junctional Adhesion Molecule-A (JAM-A), Overexpressed in Breast Ductal Carcinoma In Situ (DCIS), Reduces HER2-Positive Tumor Progression.

Breast ductal carcinoma in situ (DCIS) is clinically challenging, featuring high diagnosis rates and few targeted therapies. Expression/signaling from junctional adhesion molecule-A (JAM-A) has been linked to poor prognosis in invasive breast cancers, but its role in DCIS is unknown. Since progression from DCIS to invasive cancer has been linked with overexpression of the human epidermal growth factor receptor-2 (HER2), and JAM-A regulates HER2 expression, we evaluated JAM-A as a therapeutic target in DCIS. JAM-A expression was immunohistochemically assessed in patient DCIS tissues. A novel JAM-A antagonist (JBS2) was designed and tested alone/in combination with the HER2 kinase inhibitor lapatinib, using SUM-225 cells in vitro and in vivo as validated DCIS models. Murine tumors were proteomically analyzed. JAM-A expression was moderate/high in 96% of DCIS patient tissues, versus 23% of normal adjacent tissues. JBS2 bound to recombinant JAM-A, inhibiting cell viability in SUM-225 cells and a primary DCIS culture in vitro and in a chick embryo xenograft model. JBS2 reduced tumor progression in in vivo models of SUM-225 cells engrafted into mammary fat pads or directly injected into the mammary ducts of NOD-SCID mice. Preliminary proteomic analysis revealed alterations in angiogenic and apoptotic pathways. High JAM-A expression in aggressive DCIS lesions and their sensitivity to treatment by a novel JAM-A antagonist support the viability of testing JAM-A as a novel therapeutic target in DCIS.

A Transcriptional Link between HER2, JAM-A and FOXA1 in Breast Cancer.

Overexpression of the human epidermal growth factor receptor-2 (HER2) is associated with aggressive disease in breast and certain other cancers. At a cellular level, the adhesion protein Junctional Adhesion Molecule-A (JAM-A) has been reported to regulate the expression of HER3 via a transcriptional pathway involving FOXA1. Since FOXA1 is also a suggested transcription factor for HER2, this study set out to determine if JAM-A regulates HER2 expression via a similar mechanism. An integrated tripartite approach was taken, involving cellular expression studies after targeted disruption of individual players in the putative pathway, in silico identification of relevant HER2 promoter regions and, finally, interrogation of cancer patient survival databases to deconstruct functionally important links between HER2, JAM-A and FOXA1 gene expression. The outcome of these investigations revealed a unidirectional pathway in which JAM-A expression transcriptionally regulates that of HER2 by influencing the binding of FOXA1 to a specific site in the HER2 gene promoter. Moreover, a correlation between JAM-A and HER2 gene expression was identified in 75% of a sample of 40 cancer types from The Cancer Genome Atlas, and coincident high mean mRNA expression of JAM-A, HER2 and FOXA1 was associated with poorer survival outcomes in HER2-positive (but not HER2-negative) patients with either breast or gastric tumors. These investigations provide the first evidence of a transcriptional pathway linking JAM-A, HER2 and FOXA1 in cancer settings, and support potential future pharmacological targeting of JAM-A as an upstream regulator of HER2.

Cyclophilin A regulates secretion of tumour-derived extracellular vesicles.

Extracellular Vesicles (EVs) are a heterogenous population of particles that play an important role in cell-cell communication in physiological and pathophysiological situations. In this study we reveal that the peptidyl prolyl isomerase Cyclophilin A (CypA) is enriched in cancer-derived EVs from a range of haematopoietic malignancies. CypA-enriched blood cancer EVs were taken up by normal monocytes independent of EV surface trypsin-sensitive proteins and potently stimulated pro-inflammatory MMP9 and IL-6 secretion. Further characterisation revealed that CypA is intravesicular, however, it is not present in all EVs derived from the haematopoietic cells, instead, it is predominantly located in high density EVs with a range of 1.15-1.18 g/ml. Furthermore, loss of CypA expression in haematological cancer cells attenuates high density EV-induced pro-inflammatory MMP9 and IL-6 secretion from monocytes. Mechanistically, we reveal that homozygous loss or siRNA knockdown of CypA expression significantly reduced the secretion of EVs in the range of 100-200 nm from blood cancer cells under normal and hypoxic conditions. Overall, this work reveals a novel role for CypA in cancer cell EV biogenesis.

Human Epidermal Growth Factor Receptor-3 Expression Is Regulated at Transcriptional Level in Breast Cancer Settings by Junctional Adhesion Molecule-A via a Pathway Involving Beta-Catenin and FOXA1.

The success of breast cancer therapies targeting the human epidermal growth factor receptor-2 (HER2) is limited by the development of drug resistance by mechanisms including upregulation of HER3. Having reported that HER2 expression and resistance to HER2-targeted therapies can be regulated by Junctional Adhesion Molecule-A (JAM-A), this study investigated if JAM-A regulates HER3 expression. Expressional alteration of JAM-A in breast cancer cells was used to test expressional effects on HER3 and its effectors, alongside associated functional behaviors, in vitro and semi-in vivo. HER3 transcription factors were identified and tested for regulation by JAM-A. Finally a patient tissue microarray was used to interrogate connections between putative pathway components connecting JAM-A and HER3. This study reveals for the first time that HER3 and its effectors are regulated at gene/protein expression level by JAM-A in breast cancer cell lines; with functional consequences in in vitro and semi-in vivo models. In bioinformatic, cellular and patient tissue models, this was associated with regulation of the HER3 transcription factor FOXA1 by JAM-A via a pathway involving ß-catenin. Our data suggest a novel model whereby JAM-A expression regulates ß-catenin localization, in turn regulating FOXA1 expression, which could drive HER3 gene transcription. JAM-A merits investigation as a novel target to prevent upregulation of HER3 during the development of resistance to HER2-targeted therapies, or to reduce HER3-dependent tumorigenic signaling.

ADAM22/LGI1 complex as a new actionable target for breast cancer brain metastasis.

BACKGROUND: Metastatic breast cancer is a major cause of cancer-related deaths in woman. Brain metastasis is a common and devastating site of relapse for several breast cancer molecular subtypes, including oestrogen receptor-positive disease, with life expectancy of less than a year. While efforts have been devoted to developing therapeutics for extra-cranial metastasis, drug penetration of blood-brain barrier (BBB) remains a major clinical challenge. Defining molecular alterations in breast cancer brain metastasis enables the identification of novel actionable targets. METHODS: Global transcriptomic analysis of matched primary and metastatic patient tumours (n = 35 patients, 70 tumour samples) identified a putative new actionable target for advanced breast cancer which was further validated in vivo and in breast cancer patient tumour tissue (n = 843 patients). A peptide mimetic of the target's natural ligand was designed in silico and its efficacy assessed in in vitro, ex vivo and in vivo models of breast cancer metastasis. RESULTS: Bioinformatic analysis of over-represented pathways in metastatic breast cancer identified ADAM22 as a top ranked member of the ECM-related druggable genome specific to brain metastases. ADAM22 was validated as an actionable target in in vitro, ex vivo and in patient tumour tissue (n = 843 patients). A peptide mimetic of the ADAM22 ligand LGI1, LGI1MIM, was designed in silico. The efficacy of LGI1MIM and its ability to penetrate the BBB were assessed in vitro, ex vivo and in brain metastasis BBB 3D biometric biohybrid models, respectively. Treatment with LGI1MIM in vivo inhibited disease progression, in particular the development of brain metastasis. CONCLUSION: ADAM22 expression in advanced breast cancer supports development of breast cancer brain metastasis. Targeting ADAM22 with a peptide mimetic LGI1MIM represents a new therapeutic option to treat metastatic brain disease.

Spatially resolved diffuse imaging for high-speed depth estimation of jet injection.

We investigate the use of spatially resolved diffuse imaging to track a fluid jet delivered at high speed into skin tissue. A jet injector with a short needle to deliver drugs beneath the dermis, is modified to incorporate a laser beam into the jet, which is ejected into ex vivo porcine tissue. The diffuse light emitted from the side and top of the tissue sample is recorded using high-speed videography. Similar experiments, using a depth-controlled fiber optic source, generate a reference dataset. The side light distribution is related to source depth for the controlled-source experiments and used to track the effective source depth of the injections. Postinjection X-ray images show agreement between the jet penetration and ultimate light source depth. The surface light intensity profile is parameterized with a single parameter and an exponential function is used to relate this parameter to source depth for the controlled-source data. This empirical model is then used to estimate the effective source depth from the surface profile of the injection experiments. The depth estimates for injections into fat remain close to the side depth estimates, with a root-mean-square error of 1.1 mm, up to a source depth of 8 mm.

Classification of diffuse light emission profiles for distinguishing skin layer penetration of a needle-free jet injection.

In this work, a system is developed for tracking the skin layer to which a needle-free jet injection of fluid has penetrated by incorporating a laser beam into the jet, and measuring the diffuse light emitted from skin tissue. Monitoring the injection in this way offers the ability to improve the reliability of drug delivery with this transdermal delivery method. A laser beam, axially aligned with a jet of fluid, created a distribution of diffuse light around the injection site that varied as the injection progressed. High-speed videography was used to capture the diffuse light emission from laser-coupled jet injections into samples of porcine skin, fat, and muscle. The injection produced a distribution of diffuse light around the injection site that varied as the injection descended. A classifier, trained to distinguish whether the light source was located in the fat or muscle from surface intensity profile measurements, correctly identified the injected layer in 97.2 % of the cases when cross-examined against estimates using the light distribution emitted from the side of the sample.

High-speed X-ray analysis of liquid delivery during jet injection.

The effect of varying velocity during jet injection on the dispersion of fluid into tissue is investigated using a custom-built X-ray imaging system. Injections are performed into ex-vivo porcine abdominal tissue using a voice coil actuated injection device. Single velocity and two-phase velocity injections reveal the complex nature of the dispersion of the fluid jet in layered tissue and highlight the effects of changing the jet velocity following the initial penetration of the liquid into the tissue.

Recessive NEK9 mutation causes a lethal skeletal dysplasia with evidence of cell cycle and ciliary defects.

Skeletal dysplasias are a clinically and genetically heterogeneous group of bone and cartilage disorders. Whilst >450 skeletal dysplasias have been reported, 30% are genetically uncharacterized. We report two Irish Traveller families with a previously undescribed lethal skeletal dysplasia characterized by fetal akinesia, shortening of all long bones, multiple contractures, rib anomalies, thoracic dysplasia, pulmonary hypoplasia and protruding abdomen. Single nucleotide polymorphism homozygosity mapping and whole exome sequencing identified a novel homozygous stop-gain mutation in NEK9 (c.1489C>T; p.Arg497*) as the cause of this disorder. NEK9 encodes a never in mitosis gene A-related kinase involved in regulating spindle organization, chromosome alignment, cytokinesis and cell cycle progression. This is the first disorder to be associated with NEK9 in humans. Analysis of NEK9 protein expression and localization in patient fibroblasts showed complete loss of full-length NEK9 (107 kDa). Functional characterization of patient fibroblasts showed a significant reduction in cell proliferation and a delay in cell cycle progression. We also provide evidence to support possible ciliary associations for NEK9. Firstly, patient fibroblasts displayed a significant reduction in cilia number and length. Secondly, we show that the NEK9 orthologue in Caenorhabditis elegans, nekl-1, is almost exclusively expressed in a subset of ciliated cells, a strong indicator of cilia-related functions. In summary, we report the clinical and molecular characterization of a lethal skeletal dysplasia caused by NEK9 mutation and suggest that this disorder may represent a novel ciliopathy.

Light source depth estimation in porcine skin using spatially resolved diffuse imaging.

We present an inexpensive imaging system for measuring the diffuse surface radiance profile produced by a light source within a turbid medium. The diffusion model of light propagation in multiple scattering media is used to estimate the optical properties of a sample and subsequently approximate the depth of an optical source. The system is shown to accurately estimate the relative changes in source depth in a homogeneous phantom. The absolute depth estimate may be improved with a better estimate of the optical parameters. Preliminary tests on a porcine skin sample show that the simple model can be used to roughly track the relative changes in the depth of a source in a layered medium. However, a rigorous model of the layered geometry may be required to more accurately localize a source, particularly near interfaces between tissue layers.

Breast cancer cell migration is regulated through junctional adhesion molecule-A-mediated activation of Rap1 GTPase.

INTRODUCTION: The adhesion protein junctional adhesion molecule-A (JAM-A) regulates epithelial cell morphology and migration, and its over-expression has recently been linked with increased risk of metastasis in breast cancer patients. As cell migration is an early requirement for tumor metastasis, we sought to identify the JAM-A signalling events regulating migration in breast cancer cells. METHODS: MCF7 breast cancer cells (which express high endogenous levels of JAM-A) and primary cultures from breast cancer patients were used for this study. JAM-A was knocked down in MCF7 cells using siRNA to determine the consequences for cell adhesion, cell migration and the protein expression of various integrin subunits. As we had previously demonstrated a link between the expression of JAM-A and ß1-integrin, we examined activation of the ß1-integrin regulator Rap1 GTPase in response to JAM-A knockdown or functional antagonism. To test whether JAM-A, Rap1 and ß1-integrin lie in a linear pathway, we tested functional inhibitors of all three proteins separately or together in migration assays. Finally we performed immunoprecipitations in MCF7 cells and primary breast cells to determine the binding partners connecting JAM-A to Rap1 activation. RESULTS: JAM-A knockdown in MCF7 breast cancer cells reduced adhesion to, and migration through, the ß1-integrin substrate fibronectin. This was accompanied by reduced protein expression of ß1-integrin and its binding partners αV- and α5-integrin. Rap1 activity was reduced in response to JAM-A knockdown or inhibition, and pharmacological inhibition of Rap1 reduced MCF7 cell migration. No additive anti-migratory effect was observed in response to simultaneous inhibition of JAM-A, Rap1 and ß1-integrin, suggesting that they lie in a linear migratory pathway. Finally, in an attempt to elucidate the binding partners putatively linking JAM-A to Rap1 activation, we have demonstrated the formation of a complex between JAM-A, AF-6 and the Rap1 activator PDZ-GEF2 in MCF7 cells and in primary cultures from breast cancer patients. CONCLUSIONS: Our findings provide compelling evidence of a novel role for JAM-A in driving breast cancer cell migration via activation of Rap1 GTPase and ß1-integrin. We speculate that JAM-A over-expression in some breast cancer patients may represent a novel therapeutic target to reduce the likelihood of metastasis.

Tight junctions: a barrier to the initiation and progression of breast cancer?

Breast cancer is a complex and heterogeneous disease that arises from epithelial cells lining the breast ducts and lobules. Correct adhesion between adjacent epithelial cells is important in determining the normal structure and function of epithelial tissues, and there is accumulating evidence that dysregulated cell-cell adhesion is associated with many cancers. This review will focus on one cell-cell adhesion complex, the tight junction (TJ), and summarize recent evidence that TJs may participate in breast cancer development or progression. We will first outline the protein composition of TJs and discuss the functions of the TJ complex. Secondly we will examine how alterations in these functions might facilitate breast cancer initiation or progression; by focussing on the regulatory influence of TJs on cell polarity, cell fate and cell migration. Finally we will outline how pharmacological targeting of TJ proteins may be useful in limiting breast cancer progression. Overall we hope to illustrate that the relationship between TJ alterations and breast cancer is a complex one; but that this area offers promise in uncovering fundamental mechanisms linked to breast cancer progression.

Clinical response of two brush-applied peroxide whitening systems.

OBJECTIVE: A randomized, examiner-blind parallel design study was conducted to evaluate the clinical efficacy and tolerability of two brush-applied, peroxide-based tooth whiteners. METHODOLOGY: A total of 38 subjects were randomized to Colgate Simply White, an 18% carbamide peroxide paint-on liquid in an applicator bottle, or Crest Night Effects, a 19% sodium percarbonate system in unit dose sachets that dries to form an adherent film. Treatment was for 14 days. The 18% carbamide peroxide gel was applied twice daily, while the 19% sodium percarbonate film was applied for overnight use. Subjects were evaluated at baseline, and again after 7 and 14 days of treatment using digital images to assess tooth color, and oral examinations to establish safety. Whitening efficacy was determined by evaluating the reduction in tooth yellowness (delta b*), increase in tooth brightness (delta L*), reduction in tooth redness (delta a*) and overall tooth color change relative to pure white (delta W*). RESULTS: After 14 days, the mean change in yellowness (delta b*) was -1.43 +/- 0.20 for the 19% sodium percarbonate film, and -0.44 +/- 0.06 for the 18% carbamide peroxide gel. Mean change in brightness (delta L*) was 1.13 +/- 0.13 and 0.55 +/- 0.13 in the film and gel groups, respectively. Between-group comparisons showed highly significant (p < or = 0.003) whitening for the 19% sodium percarbonate film compared to the 18% carbamide peroxide gel for all 14-day color measurements (delta b*, delta L*, delta a* or delta W). Both products were well-tolerated, with no subjects discontinuing treatment early due to a causal adverse event. CONCLUSION: In a direct clinical comparison of two brush-applied products over 14 days, Crest Night Effects provided significant and meaningful improvement in tooth color, representing more than double the whitening measured for the Colgate Simply White.

Experimental gingivitis studies: effects of triclosan and triclosan-containing dentifrices on dental plaque and gingivitis in three-week randomized controlled clinical trials.

A recently reported six-month gingivitis study demonstrated that in subjects with gingivitis, a triclosan/pyrophosphate dentifrice provided supragingival plaque control. The level of plaque reduction was comparable with that reported for other triclosan-containing dentifrices; however, no reductions in gingivitis were observed for triclosan/pyrophosphate relative to the negative control. One possible explanation of this result is that the Hawthorne effect in the study was too great to allow the detection of a treatment benefit for the triclosan product. In order to further explore the relevance of these results, three independent clinical studies were undertaken utilizing designs based on a 21-day experimental gingivitis model in which Hawthorne effects are minimized, in part due to the absence of toothbrushing. In each model, a pre-study prophylaxis was followed by a three-week period of oral hygiene instruction to establish optimum baseline gingival health in study participants. The studies varied in enrollment; 120, 33 and 32 subjects completed treatment on studies 1, 2, and 3, respectively. In study 1, test articles were dentifrice products (0.28% triclosan/5% pyrophosphate/0.145% sodium fluoride, 0.2% triclosan/0.5% zinc citrate/0.112% sodium fluoride, 0.145% sodium fluoride and 0.15% sodium monofluorophosphate) applied neat and undiluted via a performed tooth shield (that prevents mechanical tooth-brushing at the test sites in the oral cavity) in a partial mouth design. In study 2, test articles were also dentifrice products (0.28% triclosan/5% pyrophosphate/0.243% sodium fluoride, 0.3% triclosan/2% Gantrez copolymer/0.24% sodium fluoride and 0.243% sodium fluoride) but administered to subjects in the form of 1:3 aqueous slurry rinses. Lastly, in study 3, test articles were all mouthrinses (0.12% chlorhexidine, 0.045% triclosan in ethanol plus respective vehicle placebos). Clinical assessments to quantify the test articles' effects on the development of plaque and gingivitis were conducted at baseline (studies 1, 2 and 3), day 7 (studies 2 and 3), day 14 (studies 2 and 3) and day 21 (studies 1, 2 and 3). In study 1, no statistically significant treatment effects were observed between the test articles and controls for plaque or gingivitis development. In study 2, no statistically significant treatment effects were observed at any time point between test products for the development of gingivitis. At days 7 and 14, there were no significant differences between test products and control for plaque development as well. At day 21, the group rinsing with the triclosan/pyrophosphate/sodium fluoride slurry had significantly less plaque accumulation than the group rinsing with the triclosan/copolymer/sodium fluoride slurry (p < 0.05); however, neither of the groups using test products containing triclosan was significantly different for plaque development from the group using the sodium fluoride control test article. In addition, aspartate aminotransferase activity in gingival crevicular fluid was assayed at days 0 and 21; no between-group differences were found at either of these time points, though day 21 AST activities were higher than those at baseline. In study 3, statistically significant treatment differences in plaque regrowth and gingivitis were observed at day 21 for the chlorhexidine rinse versus all other rinses (p < 0.05). No other statistically significant treatment effects were observed between test compounds at any other time points. The results benchmark the anti-plaque and anti-gingivitis benefit for a range of triclosan-based product forms against positive and negative controls in a three different experimental gingivitis models, a design considered predictive of clinical efficacy in longer-term investigations. It is concluded that dentifrice products containing triclosan do not possess sufficient antimicrobial activity to suppress plaque and gingivitis development in the absence of normal oral hygiene, and that relative to chlorhexidine, triclosan itself offers only modest efficacy for the prevention of plaque accumulation and therefore the delayed onset of gingivitis.