HPV-Related Oropharyngeal Cancer and Biomarkers Based on Epigenetics and Microbiome Profile.

H uman papillomavirus (HPV) is considered the main cause of the increasing incidence rates of oropharyngeal squamous cell carcinoma (OPSCC), and soon, the global burden of HPV-related OPSCC is predicted to exceed that of cervical cancer. Moreover, a different molecular profile for HPV-related OPSCC has been described, opening new promising targeted therapies and immunotherapy approaches. Epigenetic and microbiome-based exploration of biomarkers has gained growing interest with a view to the primary oropharyngeal cancer (OPC) screening. Understanding the role of the epigenetic mechanism and the changes that occur during pathogenesis shows appreciable progress in recent years. The different methylation status of DNA and miRNAs demonstrates the value of possible biomarkers discriminating even in different stages of dysplasia. Through whole-genome bisulfite sequencing, differentially methylated regions (DMRs) hold the key to recover missing information. O n the other hand, the microbiota investigation signifies a new biomarker approach for the evaluation of OPC. Along with known cofactors playing a major role in microbiota differentiation, HPV-related cases must be explored further for better understanding. The dynamic approach of the shotgun metagenomic sequencing will robustly fill the gap especially in species/strain level and consequently to biomarker detection. The constantly growing incidence of HPV-related OPC should lead us in further investigation and understanding of the unique features of the disease, more accurate diagnostic methods, along with the development and implementation of new, targeted therapies. This paper comprehensively reviews the significance of biomarkers based on epigenetics and microbiome profile in the accuracy of the diagnosis of the HPV-related cancer in the oropharynx.

Development of an inductively coupled MR coil system for imaging and spectroscopic analysis of an implantable bioartificial construct at 11.1 T.

Developing a method to noninvasively monitor tissue-engineered constructs is critical for the optimization of construct design and for assessing therapeutic efficacy. For this purpose, NMR is a powerful technique that can be used to obtain both images and spectroscopic data. But the inherent sensitivity of NMR limits the observation of a bioartificial construct with current NMR surface coil technology. In this study, we address this limitation through the development of an inductively coupled, implantable coil system, demonstrate its use at high field (11.1 T), and investigate the use of this coil system for monitoring a bioartificial construct in vitro and in vivo. The results establish that large gains in signal to noise can be obtained with this coil system over that obtainable with a surface coil. This coil system provides a means to quantitatively analyze the structure and function of implanted bioartificial organs.

Use of magnetic nanoparticles to monitor alginate-encapsulated betaTC-tet cells.

Noninvasive monitoring of tissue-engineered constructs is an important component in optimizing construct design and assessing therapeutic efficacy. In recent years, cellular and molecular imaging initiatives have spurred the use of iron oxide-based contrast agents in the field of NMR imaging. Although their use in medical research has been widespread, their application in tissue engineering has been limited. In this study, the utility of monocrystalline iron oxide nanoparticles (MIONs) as an NMR contrast agent was evaluated for betaTC-tet cells encapsulated within alginate/poly-L-lysine/alginate (APA) microbeads. The constructs were labeled with MIONs in two different ways: 1) MION-labeled betaTC-tet cells were encapsulated in APA beads (i.e., intracellular compartment), and 2) MION particles were suspended in the alginate solution prior to encapsulation so that the alginate matrix was labeled with MIONs instead of the cells (i.e., extracellular compartment). The data show that although the location of cells can be identified within APA beads, cell growth or rearrangement within these constructs cannot be effectively monitored, regardless of the location of MION compartmentalization. The advantages and disadvantages of these techniques and their potential use in tissue engineering are discussed.

Nasal chondromesenchymal hamartoma in a child.

Nasal chondromesenchymal hamartoma (NCMH) is a benign tumor that was described in 1998. The occurrence of this lesion in the nasal cavity of infants and children is especially rare, with only 21 cases reported in the international literature. We report a 12-month-old boy with respiratory distress due to nasal obstruction. Computed tomographic scan and magnetic resonance imaging examination demonstrated a soft-tissue mass obstructing the left nasal cavity. Digital subtraction angiography and preoperative superselective embolization with microparticles were also performed. The tumor was completely resected surgically. Histopathology and immunohistochemical analyses of the tumor disclosed a NCMH. The imaging characteristics of the tumor are described and the radiology literature is reviewed.

Novel synthesis of cerium oxide nanoparticles for free radical scavenging.

AIMS: The aim of this article is to present a novel synthetic route to form CeO(2) nanoparticles that protects against the detrimental influence of oxidative stress in mammalian cells. METHODS: The noncytotoxic surfactant lecithin was used to synthesize CeO(2) nanoparticles and the products were colloidally stabilized in a biocompatible tri-sodium citrate buffer. These nanoparticles were delivered into murine insulinoma betaTC-tet cells, and intracellular free radical concentrations responding to exposure to hydroquinone were measured in a variety of extracellular CeO(2) concentrations. RESULTS: Well-dispersed, highly crystallized CeO(2) nanoparticles of 3.7 nm in size were achieved that are chemically and colloidally stable in Dulbecco's modified Eagle's medium for extended periods of time. Treating betaTC-tet cells with these nanoparticles alleviated detrimental intracellular free radical levels down to the primary level. CONCLUSION: CeO(2) nanoparticles synthesized from this route are demonstrated to be effective free radical scavengers within betaTC-tet cells. Furthermore, it is shown that CeO(2) nanoparticles provide an effective means to improve cellular survival in settings wherein cell loss due to oxidative stress limits native function.

13C NMR isotopomeric analysis and its application in the study of endocrine cell metabolism and function.

Defining mechanisms and enzymatic paths critical to cellular function (e.g., secretion) of endocrine cells is a key research goal that can lead toward novel avenues of therapeutic intervention for a variety of disorders. 13C NMR spectroscopy and isotopomer analysis of cell extracts are excellent tools to quantitatively assess metabolism through intermediate labeling and estimate carbon entry to the TCA cycle. Discussed are: cell lines and in vitro culturing; extraction of intracellular material; NMR spectroscopy of the extract; isotopomeric analysis and modeling to obtain relative metabolic fluxes to the TCA cycle. This paper describes issues related to the application of NMR spectroscopic techniques on cell line extracts. Included are results of two studies that illustrate considerations that must be taken when performing analogous studies on neuroendocrine tissue: one involving the effect of media composition on cell behavior and isotopomer labeling; the second looking at effects of applying different metabolic models to 13C data and inferences that may be drawn. NMR isotopomeric analysis is a powerful technique that may be applied to better understand endocrine cell function.

Non-invasive evaluation of alginate/poly-l-lysine/alginate microcapsules by magnetic resonance microscopy.

In this report, we present data to demonstrate the utility of (1)H MR microscopy to non-invasively examine alginate/poly-l-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly-l-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6+/-6.2 microm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 min). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T(2) relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T(2) over the duration of the experiment, while ADC was unaffected. This decrease in T(2) is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T(2) was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T(2) or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions.

Magnetic resonance spectroscopic investigation of mitochondrial fuel metabolism and energetics in cultured human fibroblasts: effects of pyruvate dehydrogenase complex deficiency and dichloroacetate.

The pyruvate dehydrogenase complex (PDC) is integral to metabolism and energetics. Congenital PDC deficiency leads to lactic acidosis, neurological degeneration and early death. An investigational compound for such defects is dichloroacetate (DCA), which activates the PDC (inhibiting reversible phosphorylation of the E1alpha subunit) and decreases its turnover. Here, primary human fibroblast cultures from five healthy subjects and six patients with mutations in the PDC-E1 component were grown in media+/-DCA, exposed to media containing (13)C-labeled glucose, and studied (as cell extracts) by nuclear magnetic resonance (NMR) spectroscopy. Computer modeling of NMR-derived (13)C-glutamate isotopomeric patterns estimated relative carbon flow through TCA cycle-associated pathways and characterized effects of PDC deficiency on metabolism and energetics. Rates of glucose consumption (GCR) and lactate production (LPR) were measured. With the exception of one patient cell line expressing an unusual splicing mutation, PDC-deficient cells had significantly higher GCR, LPR and label-derived acetyl-CoA, indicative of increased glycolysis vs. controls. In all cells, DCA caused a major shift (40% decrease) from anaplerotic-related pathways (e.g., pyruvate carboxylase) toward flux through PDC. Ignoring the patient with the splicing mutation, DCA decreased average glycolysis (29%) in patient cells, but had no significant effect on control cells, and did not change LPR or the nucleoside triphosphate to diphosphate ratio (NTP/NDP) in either cell type. Maintenance of NTP despite reduced glycolysis indicates that DCA improves metabolic efficiency by increasing glucose oxidation. This study demonstrates that NMR spectroscopy provides insight into biochemical consequences of PDC deficiency and the mechanism of putative therapeutic agents.

Immune responses to an encapsulated allogeneic islet beta-cell line in diabetic NOD mice.

Our goal is to develop effective islet grafts for treating type 1 diabetes. Since human islets are scarce, we evaluated the efficacy of a microencapsulated insulin-secreting conditionally transformed allogeneic beta-cell line (betaTC-tet) in non-obese diabetic mice treated with tetracycline to inhibit cell growth. Relatively low serum levels of tetracycline controlled proliferation of betaTC-tet cells without inhibiting effective control of hyperglycemia in recipients. There was no significant host cellular reaction to the allografts or host cell adherence to microcapsules, and host cytokine levels were similar to those of sham-operated controls. We conclude that encapsulated allogeneic beta-cell lines may be clinically relevant, because they effectively restore euglycemia and do not elicit a strong cellular immune response following transplantation. To our knowledge, this is the first extensive characterization of the kinetics of host cellular and cytokine responses to an encapsulated islet cell line in an animal model of type 1 diabetes.

Biochemical consequences of alginate encapsulation: a NMR study of insulin-secreting cells.

In this study we explore the biochemical consequences of alginate encapsulation on betaTC3 cells. (13)C NMR spectroscopy and isotopomer analysis were used to investigate the effects of encapsulation on several enzymatic processes associated with the TCA cycle. Our data show statistically significant differences in various enzymatic fluxes related to the TCA cycle and insulin secretion between monolayer and alginate-encapsulated cultures. The principal cause for these effects was the process of trypsinization. Embedding the trypsinized cells in alginate beads did not have a compounded effect on the enzymatic fluxes of entrapped cells. However, an additional small but statistically significant decrease in insulin secretion was measured in encapsulated cells. Finally, differences in either enzymatic fluxes or glucose consumption as a function of bead diameter were not observed. However, differences in T(2), assessed by (1)H NMR microimaging, were observed as a function of bead diameter, suggesting that smaller beads became more organized with time in culture, while larger beads displayed a looser organization.

Insulin secretion dynamics of free and alginate-encapsulated insulinoma cells.

This study investigates the effect of alginate/poly-L: -lysine/alginate (APA) encapsulation on the insulin secretion dynamics exhibited by an encapsulated cell system. Experiments were performed with the aid of a home-built perfusion apparatus providing a 1 min temporal resolution. Insulin profiles were measured from: (i) murine insulinoma betaTC3 cells encapsulated in calcium alginate/poly-L: -lysine/alginate (APA) beads generated with high guluronic (G) or high mannuoric (M) content alginate, and (ii) murine insulinoma betaTC-tet cells encapsulated in high M APA beads and propagated in the presence and absence of tetracycline. Results show that encapsulation in APA beads did not affect the insulin secretion profile shortly post-encapsulation. However, remodeling of the beads due to cell proliferation affected the insulin secretion profiles; and inhibiting remodeling by suppressing cell growth preserved the secretion profile. The implications of these findings regarding the in vivo function of encapsulated insulin secreting cells are discussed.

Effects of growth regulation on conditionally-transformed alginate-entrapped insulin secreting cell lines in vitro.

The ability to control cell growth is an issue of critical importance for the use of transformed beta-cell lines within a bioartificial pancreas. Such control can be achieved either by entrapping the cells in a biomaterial that can inhibit cell proliferation or by genetically modifying the cells to regulate growth. Integrating tetracycline-off or -on operon systems into murine insulinoma cell lines (betaTC-tet and R7T1, respectively) allows cell growth regulation upon exposure to tetracycline (TC) or its derivative doxycycline (Dox), respectively. However, the effects of this regulatory approach on the long-term phenotypic metabolic and secretory stability of alginate-entrapped cells have yet to be thoroughly investigated. In this study, cultures of betaTC-tet and R7T1 cells entrapped in alginate beads were allowed to grow freely, or were growth-regulated, either at the onset, or after 20 days of growth. The data show that growth regulation of alginate-entrapped cells is achievable with chronic administration of the regulatory compound in a concentration-dependent manner. However, as these cultures age, the amount of insulin released does not always reflect the metabolic and histological characteristics of the cultures. This change, coupled with a loss of glucose stimulated insulin secretion in the Dox treated R7T1 cell line, indicate a phenotypic shift of cells with an activated tet-operon. These observations have implications on the selection and long-term function of three-dimensional bioartificial pancreatic constructs that include conditionally transformed beta-cell lines.

Magnetically labeled insulin-secreting cells.

Iron oxide nanoparticles have been shown to magnetically label cells in order to visualize them in vivo via MR imaging. This technology has yet to be implemented in insulin secreting cells, thus it is not known whether the presence of these nanoparticles in the cytoplasm of the cells affects insulin secretion. This study investigates the effectiveness and consequence of labeling mouse insulinoma betaTC3 and betaTC-tet cells with monocrystalline iron oxide nanoparticles (MION). Our data show that MION can be internalized in both betaTC3 and betaTC-tet cells following a 24h exposure to 0.02mg/ml MION solution. The metabolic and secretory activities of both MION-labeled cell lines were statistically indistinguishable from sham treatment. Furthermore, cell viability and apoptosis remained constant throughout the cell's exposure to MION. Finally, MR images demonstrated significant contrast between labeled and sham-treated cells. Thus, labeling murine insulinoma cell lines with magnetic iron oxide nanoparticles does not hinder their insulin secretion, while it provides MR imaging contrast.

The role of the CaCl2-guluronic acid interaction on alginate encapsulated betaTC3 cells.

Previously we demonstrated that alginate composition has a significant effect on the growth of encapsulated betaTC3 cells and consequently on the overall metabolic and secretory activities of the encapsulated cultures. Based on these results we postulated that the mechanical properties of alginate were not responsible for the observed effects but rather, changes in the strength of the alginate gel network caused by changes in the number of alginate strands held together in the "egg-box" model are responsible for the observed effects. In this study we address this hypothesis with a series of experiments in which the strength of this interaction is manipulated by varying the calcium concentration either at the time of gelation or during culture maintenance. Our data show that increasing the concentration of the CaCl2 solution used at the time of gelation, thus increasing the strength of the alginate gel network, impedes the growth characteristics of betaTC3 cells encapsulated in a high guluronic acid content alginate. This effect is amplified by maintaining a constant supply of calcium ions during culture thus sustaining the interaction between guluronic acid residues and calcium ions. However, preparations of betaTC3 cells encapsulated in an alginate with high mannuronic acid content are not affected by changes in CaCl2 concentration due to the low percentage of consecutive guluronic acid residues. Therefore, the present data show that the strength of the alginate gel network is an important factor that influences the growth characteristics of encapsulated cell preparations.

NMR properties of alginate microbeads.

Alginates are a family of unbranched polysaccharides with properties that vary widely depending on their composition. In the presence of multivalent cations (frequently Ca2+), alginates form a gel. Consequently, alginates have been used to encapsulate a variety of biological materials, including cells. In this study, we present NMR relaxation and diffusion data from alginate microbeads with similar size and properties to those used in the development of a bioartificial pancreas. Our data demonstrate that the transverse relaxation time (T2) of water within the gel depends on the guluronic acid content of the alginate, whereas the longitudinal relaxation time (T1) and the apparent diffusion coefficient of water do not. Our data further suggest that the diffusion of Ca2+ ions is hindered by the presence of a poly-L-lysine layer, a layer commonly added to provide mechanical support to the beads and immunoprotection to the encapsulated cells in the event of implantation. The impact of these data on our understanding of the role of alginate gels in the development of a bioartificial pancreas is discussed.