Tissue Of Interest Research Articles

MechanoBase: a comprehensive database for the mechanics of tissues and cells.

Mechanical aspects of tissues and cells critically influence a myriad of biological processes and can substantially alter the course of diverse diseases. The emergence of diverse methodologies adapted from physical science now permits the precise quantification of the cellular forces and the mechanical properties of tissues and cells. Despite the rising interest in tissue and cellular mechanics across fields like biology, bioengineering and medicine, there remains a noticeable absence of a comprehensive and readily accessible repository of this pertinent information. To fill this gap, we present MechanoBase, a comprehensive tissue and cellular mechanics database, curating 57 480 records from 5634 PubMed articles. The records archived in MechanoBase encompass a range of mechanical properties and forces, such as modulus and tractions, which have been measured utilizing various technical approaches. These measurements span hundreds of biosamples across more than 400 species studied under diverse conditions. Aiming for broad applicability, we design MechanoBase with user-friendly search, browsing and data download features, making it a versatile tool for exploring biomechanical attributes in various biological contexts. Moreover, we add complementary resources, including the principles of popular techniques, the concepts of mechanobiology terms and the cellular and tissue-level expression of related genes, offering scientists unprecedented access to a wealth of knowledge in this field of research. Database URL: https://zhanglab-web.tongji.edu.cn/mechanobase/ and https://compbio-zhanglab.org/mechanobase/.

A revised mathematical solution to the Penne bioheat equation for deep rooted tissues using the variable separation method

The temperature distribution of tissues under the skin surface was obtained using Penne’s bio-heat equation and the separation of variables technique. The resulting solution indicated a slight temperature difference between the recorded skin temperature and the temperature of the tissue of interest (TOI), which is a direct indication of the TOI's temperature. According to the graphical analysis, temperature is proportionally related to time and tissue distance from the skin surface. As a result, the given analytic solution may be utilized to easily analyze deeprooted tissues while accounting for thermal properties.

Phenotype-tissue expression and exploration (PTEE) resource facilitates the choice of tissue for RNA-seq-based clinical genetics studies

BackgroundRNA-seq emerges as a valuable method for clinical genetics. The transcriptome is “dynamic” and tissue-specific, but typically the probed tissues to analyze (TA) are different from the tissue of interest (TI) based on pathophysiology.ResultsWe developed Phenotype-Tissue Expression and Exploration (PTEE), a tool to facilitate the decision about the most suitable TA for RNA-seq. We integrated phenotype-annotated genes, used 54 tissues from GTEx to perform correlation analyses and identify expressed genes and transcripts between TAs and TIs. We identified skeletal muscle as the most appropriate TA to inquire for cardiac arrhythmia genes and skin as a good proxy to study neurodevelopmental disorders. We also explored RNA-seq limitations and show that on-off switching of gene expression during ontogenesis or circadian rhythm can cause blind spots for RNA-seq-based analyses.ConclusionsPTEE aids the identification of tissues suitable for RNA-seq for a given pathology to increase the success rate of diagnosis and gene discovery. PTEE is freely available at https://bioinf.eva.mpg.de/PTEE/

An accelerated procedure for approaching and imaging of optically branded region of interest in tissue.

Many areas of biology have benefited from advances in light microscopy (LM). However, one limitation of the LM approach is that numerous critically important aspects of subcellular machineries are well beyond the resolution of conventional LM. For studying these, electron microscopy (EM) remains the technique of choice to visualize and identify macromolecules at the ultrastructural level. The most powerful approach is combining both techniques, LM and EM (i.e., to apply correlative light/electron microscopy, CLEM) to image exactly the same region of interest. This combination allows, for example, to immuno-localize proteins by LM and then to visualize the ultrastructural context of the same region of the sample. However, the identification and correlation of the regions of interest (ROIs) at the levels of LM and EM remains a major challenge, mostly due to the difficulties with correlation along the Z-axis for both modalities. In this chapter, we address this difficulty and describe an approach for performing CLEM in tissue samples using marks from near-infrared branding as indicators of a ROI, and then using serial block face-scanning electron microscopy (SBF-SEM) to identify and approach this ROI. Once a ROI has been approached, serial sections are collected on grids for high-resolution imaging by transmission EM, and subsequent correlation with LM images showing labeled proteins.

Unique circulating microrna profile identified in early radiographic knee osteoarthritis

Purpose: The complex pathophysiology of osteoarthritis (OA) is likely the result of a combination of local changes in the joint and systemic changes throughout the body. There are relatively few large-scale studies with clearly defined patient cohorts that have profiled molecular differences in circulating factors in OA. Next generation sequencing is considered to be the gold-standard approach for unbiased profiling of targets of interest in tissues of interest. When applied to circulating factors, this method presents the opportunity to comprehensively profile factors that may be associated with particular stages of disease, offering the sensitivity and specificity required to identify novel and low abundance transcripts.

Autoradiography as a Simple and Powerful Method for Visualization and Characterization of Pharmacological Targets.

In vitro autoradiography aims to visualize the anatomical distribution of a protein of interest in tissue from experimental animals as well as humans. The method is based on the specific binding of a radioligand to its biological target. Therefore, frozen tissue sections are incubated with radioligand solution, and the binding to the target is subsequently localized by the detection of radioactive decay, for example, by using photosensitive film or phosphor imaging plates. Resulting digital autoradiograms display remarkable spatial resolution, which enables quantification and localization of radioligand binding in distinct anatomical structures. Moreover, quantification allows for the pharmacological characterization of ligand affinity by means of dissociation constants (Kd), inhibition constants (Ki) as well as the density of binding sites (Bmax) in selected tissues. Thus, the method provides information about both target localization and ligand selectivity. Here, the technique is exemplified with autoradiographic characterization of the high-affinity γ-hydroxybutyric acid (GHB) binding sites in mammalian brain tissue, with special emphasis on methodological considerations regarding the binding assay parameters, the choice of the radioligand and the detection method.

An injectable platelet lysate-hyaluronic acid hydrogel supports cellular activities and induces chondrogenesis of encapsulated mesenchymal stem cells

Developing scaffolds that can provide cells and biological cues simultaneously in the defect site is of interest in tissue engineering field. In this study, platelet lysate (PL) as an autologous and inexpensive source of growth factors was incorporated into a cell-laden injectable hyaluronic acid-tyramine (HA-TA) hydrogel. Subsequently, the effect of platelet lysate on cell attachment, viability and differentiation of human mesenchymal stem cell (hMSCs) toward chondrocytes was investigated. HA-TA conjugates having a degree of substitution of 20 TA moieties per 100 disaccharide units were prepared and crosslinked in the presence of horseradish peroxidase and low concentrations of hydrogen peroxide. The storage moduli of the gels ranged from 500 to 2000 Pa and increased with increasing polymer concentration. In contrast to a retained round shape of the cells when using pure HA-TA hydrogel, the hMSCs attached and spread out in PL enriched matrix. The enrichment of hMSCs laden HA-TA hydrogels with PL induced a cartilage like extra cellular matrix deposition in vitro. The hMSCs increasingly deposited collagen type II and proteoglycans over time. The deposition of the new extracellular matrix (ECM) is simultaneous with gel degradation and resulted ultimately in the formation of a tough dense matrix. These findings demonstrate the potential of injectable HA-TA-PL hydrogel as a cell delivery system for cartilage regeneration. Statement of SignificanceCartilage tissue has limited ability to self-repair because of its avascular nature. To have an efficient cartilage tissue regeneration, we combined platelet lysate (PL), as an autologous and inexpensive source of growth factors, with an injectable hyaluronic acid tyramine (HA-TA) hydrogel scaffold. Platelet lysate had a vital role in supporting human mesenchymal stem cells (hMSCs) activities, like cell attachment, viability and proliferation in the 3D hydrogel structure. Also, the hMSCs encapsulated HA-TA induced hyaline cartilage generation when placed in chondrogenic differentiation medium. This study introduces a new system for cartilage tissue engineering, which can be injected in a minimally invasive manner and is rich with patient’s own growth factors and biological cues.

Identification of Foreign Particles in Human Tissues Using Raman Microscopy.

The goal of this study was to precisely and unambiguously identify foreign particles in human tissues using a combination of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicrometer spatial resolution. This identification for patient care and research has been traditionally studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction, all with some limitations. We designed a model system of stained and unstained cells that contained birefringent talc particles and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra obtained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely assigned to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micrometer in diameter. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.

<em>In Situ</em> MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled "Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo." The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

In Situ MHC-tetramer Staining and Quantitative Analysis to Determine the Location, Abundance, and Phenotype of Antigen-specific CD8 T Cells in Tissues.

T cells are critical to many immunological processes, including detecting and eliminating virus-infected cells, preventing autoimmunity, assisting in B-cell and plasma-cell production of antibodies, and detecting and eliminating cancer cells. The development of MHC-tetramer staining of antigen-specific T cells analyzed by flow cytometry has revolutionized our ability to study and understand the immunobiology of T cells. While extremely useful for determining the quantity and phenotype of antigen-specific T cells, flow cytometry cannot determine the spatial localization of antigen-specific T cells to other cells and structures in tissues, and current disaggregation techniques to extract the T cells needed for flow cytometry have limited effectiveness in non-lymphoid tissues. In situ MHC-tetramer staining (IST) is a technique to visualize T cells that are specific for antigens of interest in tissues. In combination with immunohistochemistry (IHC), IST can determine the abundance, location, and phenotype of antigen-specific CD8 and CD4 T cells in tissues. Here, we describe a protocol to stain and enumerate antigen-specific CD8 T cells, with specific phenotypes located within specific tissue compartments. These procedures are the same that we used in our recent publication by Li et al., entitled "Simian Immunodeficiency Virus-Producing Cells in Follicles Are Partially Suppressed by CD8+ Cells In Vivo." The methods described are broadly applicable because they can be used to localize, phenotype, and quantify essentially any antigen-specific CD8 T cell for which MHC tetramers are available, in any tissue.

Abstract 2064: Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft

Abstract Introduction Mass Spectrometry Imaging (MSI) is an emerging technique in preclinical analysis that allows for analysis of the distribution and the quantification of target molecules in tissue sections. The main advantage of this imaging technology is the detection of the molecule of interest in tissues without labelling and with high specificity. MSI is now routinely used for small molecule distribution analysis in lead optimization process, and in PK/PD or toxicity studies. Due to the mass range limitation of the instrument analyzer one of the main challenges is the detection of administered large molecules such as therapeutic antibodies. . In this study, we describe the detection and the distribution of trastuzumab, a monoclonal antibody that recognizes the HER2/neu receptor, in a BT474 human breast xenograft model. Experimental procedures BT474 tumor fragments are implanted in female CB.17 SCID mice. When the tumors reach 80-120 mm3, the trastuzumab treatment of 20mg/kg with eight bi-weekly intraperitoneal injections was initiated. The endpoint was fixed at day 30 post first administration. At 50 hours post 1st, 5th and 8th doses vehicle and trastuzumab treated animals were sacrificed and tumor, plasma and carcass were collected. One section of each tumor tissue was first digested with a proteolytic enzyme deposited with a dedicated device onto the tissue. After the proteolytic digestion, the selected MALDI matrix is sprayed onto the tissue and then the distribution of the Trastuzumab is determined based on its specific peptide mass fingerprint (PMF) by high resolution mass spectrometry imaging with a MALDI-FTICR instrument (SolariX FTICR 7.0T from Bruker). The images are obtained with Quantinetix software dedicated to the MSI data set. Novel Aspect We describe a process which combines MSI and bottom-up proteomics approach directly on tissue to follow the distribution of a therapeutic antibody without any labelling. Until now, mass spectrometry imaging was used to study some biomarkers' (proteins) distribution on tissue. This work describes a novel MSI application for analysis of a dosed therapeutic antibody. Data collection is ongoing. Conclusions We have adapted the use of Mass Spectrometry Imaging to follow the distribution of a non-labeled therapeutic antibody based on its peptide mass fingerprint in dosed tissues. This opens the use of this technology to other large molecules, such as therapeutic proteins or Antibody-Drug Conjugates (ADCs) in a variety of therapeutic fields (Oncology, Immunology, CNS, etc.) In addition to the distribution of the target therapeutic antibody, Mass Spectrometry Imaging could also allow the detection of other important endogenous molecules in the same or adjacent tissue section, such as specific disease markers, thus allowing further understanding of PK/PD relationships. Citation Format: David Bonnel, Chassidy Hall, Robert J. Mullin, Kathryn A. Simon, Jonathan Stauber. Mass Spectrometry Imaging of therapeutic antibodies: Distribution of unlabeled trastuzumab in CB.17 SCID mice implanted with the human breast BT474 xenograft. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2064. doi:10.1158/1538-7445.AM2014-2064

441O - Imaging Mass Spectrometry of Novel Drug in Human Tumor Specimens: Distribution of Unlabeled Drugs to Support Early Phase Clinical Trial

ABSTRACT Aim: Assessment of drug pharmacokinetics is an important component of early phase drug development. Imaging Mass Spectrometry (IMS) is an innovative technique in the preclinical study that allows for analysis of the distribution of target molecules in tumor tissues. The advantage of imaging technology is the detection of the molecule of interest in tissues without labeling. We performed tumor biopsies in patients with solid tumors who were participating in a phase I trial (NCT01813474) of olaparib. The aim of this study was to examine drug distribution in the tumor biopsy specimens by IMS, which provides a sensitive and label-free approach to imaging drugs. Methods: Patients with solid tumors received the tablet formulation of olaparib in dose escalation (200mg BID; 300mg BID) and expansion (300mg BID) cohorts. The timing of biopsies in consenting patients was during cycle 2 and/or at the time of progression. IMS was performed using an Imaging Mass Microscope (Shimadzu, Japan). The concentrations of olaparib in tissues were validated by using LC-MS/MS. Results: In total, seven tumor biopsies were performed in six patients with solid tumors; three breast cancer including one BRCA1 mutation positive, one ovarian cancer, one peritoneal cancer, one cervical cancer. One patient had biopsies performed at the time of drug administration and progression. One patient received olaparib 200mg BID; the remaining patients received olaparib 300mg BID. IMS signal levels of olaparib correlated well with the concentration of drug in tumor tissues derived from patients using LC-MS/MS, a conventional method used in pharmacokinetic studies. The distribution of Olaparib was in the tumor region and the signal level in areas of necrosis was higher than that observed in living cell areas. Conclusions: The use of IMS has allowed to follow the distribution of an unlabeled olaparib in target tissues. In addition, this technique can also allow further understanding of PK/PD relationships of olaparib in combination with other compound at clinical trial. Disclosure: All authors have declared no conflicts of interest.

Novel Antibiotic-Eluting Gelatin-Alginate Soft Tissue Adhesives for Various Wound Closing Applications

Interest in tissue adhesives as alternatives for conventional wound closing applications such as sutures and staples has increased in the last few decades due to numerous possible advantages, including less discomfort and lower cost. In the current study, the authors developed novel tissue adhesives based on gelatin and alginate, crosslinked by carbodiimide. The antibiotic drug ceftazidime was incorporated for prevention of infection. The bonding strength of the bioadhesives, their swelling ration, drug release profile and biocompatibility were studied. The high bonding strength and good biocompatibility turns these new bioadhesives into a promising alternative for use in wound closing applications.

Novel gelatin/alginate soft tissue adhesives loaded with drugs for pain management: structure and properties

Interest in tissue adhesives as alternatives for conventional wound closing applications, such as sutures and staples, has increased in the last few decades due to numerous possible advantages, including less discomfort and lower cost. Novel tissue adhesives based on gelatin, with alginate as a polymeric additive and crosslinked by carbodiimide were developed and loaded with two types of drugs for pain relief, bupivacaine and ibuprofen, in order to improve the therapeutic effect. The release of the drugs from the adhesive matrix was found to be controlled mainly by the adhesive’s characteristics, i.e. swelling and hydrophilic group concentration. The drug characteristics, i.e. hydrophilicity and electrical interactions between the drug and the polymeric components, were also found to have some effect. Incorporation of bupivacaine was found to improve the bonding strength of the adhesive due to its inert nature and the reinforcing effect of its fibrous crystals, whereas incorporation of ibuprofen was found to have an adverse effect on the bonding strength, probably due to its reaction with the other adhesive components which increased the crosslinking density. Overall, the novel drug-eluting gelatin-based bioadhesives investigated in this research, especially those loaded with bupivacaine, demonstrated a promising potential for use in wound closing applications.

Effect of crosslinking on the mechanical properties of mineralized and non‐mineralized collagen fibers

Problems associated with current bone substitutes led to an increased interest in tissue engineered surrogates with properties similar to natural bones. Commonly used materials lack the ultimate mechanical properties. In this study, we examined the influence of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) as a crosslinking agent on the strength of collagen fibers. Collagen fibers are crosslinked with EDC, or EDC/N-hydroxysulfosuccinimide (Sulfo-NHS) in water or EDC in acetone. After which fibers are mineralized using calcium chloride and potassium phosphate. The mechanical properties of the treated fibers are measured using tensile testing. In addition, the effect of crosslinking on cellular behavior was tested by culturing bone marrow mesenchymal stem cells (BMMSCs) on modified fibers. We found that the mechanical properties of non-mineralized and mineralized collagen fibers are significantly affected by the crosslinking method. The tensile strength of single fibers is greatly improved by crosslinking with EDC in acetone. BMMSCs were found to attach and spread well on modified fibers confirming biocompatibility.

Characterization of Microcirculation in Multiple Sclerosis Lesions by Dynamic Texture Parameter Analysis (DTPA)

ObjectiveTexture analysis is an alternative method to quantitatively assess MR-images. In this study, we introduce dynamic texture parameter analysis (DTPA), a novel technique to investigate the temporal evolution of texture parameters using dynamic susceptibility contrast enhanced (DSCE) imaging. Here, we aim to introduce the method and its application on enhancing lesions (EL), non-enhancing lesions (NEL) and normal appearing white matter (NAWM) in multiple sclerosis (MS).MethodsWe investigated 18 patients with MS and clinical isolated syndrome (CIS), according to the 2010 McDonald's criteria using DSCE imaging at different field strengths (1.5 and 3 Tesla). Tissues of interest (TOIs) were defined within 27 EL, 29 NEL and 37 NAWM areas after normalization and eight histogram-based texture parameter maps (TPMs) were computed. TPMs quantify the heterogeneity of the TOI. For every TOI, the average, variance, skewness, kurtosis and variance-of-the-variance statistical parameters were calculated. These TOI parameters were further analyzed using one-way ANOVA followed by multiple Wilcoxon sum rank testing corrected for multiple comparisons.ResultsTissue- and time-dependent differences were observed in the dynamics of computed texture parameters. Sixteen parameters discriminated between EL, NEL and NAWM (pAVG = 0.0005). Significant differences in the DTPA texture maps were found during inflow (52 parameters), outflow (40 parameters) and reperfusion (62 parameters). The strongest discriminators among the TPMs were observed in the variance-related parameters, while skewness and kurtosis TPMs were in general less sensitive to detect differences between the tissues.ConclusionDTPA of DSCE image time series revealed characteristic time responses for ELs, NELs and NAWM. This may be further used for a refined quantitative grading of MS lesions during their evolution from acute to chronic state. DTPA discriminates lesions beyond features of enhancement or T2-hypersignal, on a numeric scale allowing for a more subtle grading of MS-lesions.

Gelatin–alginate novel tissue adhesives and their formulation–strength effects

Interest in tissue adhesives as alternatives for conventional wound-closing applications such as sutures and staples has increased in the last few decades due to numerous possible advantages, including less discomfort and lower cost. Novel tissue adhesives based on gelatin, with alginate as a polymeric additive and crosslinked by carbodiimide, were recently developed by our research group. The effects of the formulation parameters on the adhesives’ function were investigated in the current study. We examined the effects of gelatin and alginate concentrations and their viscosities on the ability of the bioadhesives to bind soft tissues. The effect of the crosslinking agent’s concentration was studied as well. A qualitative model describing these effects in terms of adherence mechanisms was developed. Our results show that the adherence properties of our new bioadhesives are achieved by a combination of two main mechanisms: mechanical interlocking and chemical adsorption. The former mechanism is probably more dominant. The polymer’s molecular weight and concentration affect the mechanical interlocking through mobility and penetration ability, entanglement of the three-dimensional structure and crosslinking density. The crosslinking agent’s concentration as well as the polymer’s concentration affect the crosslinking density and contribute to higher strength, achieved through both the mechanical interlocking and the chemical adsorption mechanisms. Understanding the effects of the adhesives’ components and their viscosities on the bonding strength enabled us to elucidate the bonding strength mechanisms. This can lead to proper selection of the adhesive formulation and may enable tailoring the bioadhesives to the desired applications.

Abstract 653: Gliomas: methylation status of MGMT gene monitoring in plasma DNA of patients receiving chemotherapy with alkylating agents.

Abstract The protein MGMT (O6-methylguanine-DNA-methyltransferase) is responsible for the DNA repair from damage in O6 position of guanine induced by alkylating agent such as temozolomide, which represent the conventional treatment of gliomas. Silencing of the MGMT gene by epigenetic methylation of its promoter may disable this repair mechanism, thus increasing the cytotoxicity of chemotherapy. Previous studies showed that glioblastama patients, with a methylated MGMT gene promoter in tumour tissue, are more sensitive to the action of alkylating agents and experience higher survival. The aim of the study was to verify the feasibility and the utility of monitoring the state of methylation of MGMT in circulating DNA as a predictive marker of prognosis and response to treatment. Fiftyeight patients with glioma (12 with low and 46 with high grade) who underwent surgical resection and who were eligible for therapy with temozolomide, were enrolled in the study. Blood samples for the analysis of MGMT methylation status were collected before any treatment (T0) and during the treatment at the time of each magnetic resonance imaging (about every 3 months). The methylation status of the MGMT gene promoter in circulating DNA was investigated using PCR with a specific probe for the methylated sequence. The MGMT promoter methylation status in tumour tissue and in plasma was highly concordant: in the 79% of the patients the MGMT promoter was methylated both in tumour tissue and plasma, while in the 21% of patients MGMT promoter was methylated in tissue but not in plasma (Cohen's kappa coefficient = 0.75, p <0.001). The overall survivals (OS) was higher in methylated versus unmethylated MGMT promoter patients both in tumour tissue and plasma, even if with a borderline statistically significance (p = 0.06 by the log-rank test for plasma). The risk of death increased in patients with unmethylated MGMT promoter both in tumour tissue (Adjusted HR=2.23; 95%CI 0.99-5.04) and in plasma (Adjusted HR1.73; 95%CI 0.92-3.22) Finally, in all patients after one year from diagnosis we noticed a decrease of the state of methylation of MGMT promoter in plasma. At the end of follow up in the 98% of patients who presented the methylation of the MGMT promoter at T0, a complete unmethylation was observed. In conclusion the results of this study confirm the feasibility of monitoring the state of methylation of MGMT in circulating DNA in glioma patients. MGMT promoter methylation status may be a prognostic marker of clinical interest in tissue and in plasma. Furthermore the complete unmethylation of MGMT promoter in the 98% of the glioma patients after one year from diagnosis, is a potentially important finding for future researches on biological history and treatment of gliomas. Citation Format: Valentina Fiano, Morena Trevisan, Carlotta Sacerdote, Anna Castiglione, Elisa Trevisan, Rebecca Senetta, Anna Gillio Tos, Laura De Marco, Paola Cassoni, Riccardo Soffietti, Franco Merletti. Gliomas: methylation status of MGMT gene monitoring in plasma DNA of patients receiving chemotherapy with alkylating agents. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 653. doi:10.1158/1538-7445.AM2013-653

The Effect of Mechanical Constraints on Gelatin Samples under Pulsatile Flux

It is of great interest in tissue engineering the role of collagen gel-based structures (scaffolds, grafts and-by cell seeded and maturation-tissue equivalents (TEs) for several purposes). It is expected the appropriate biological compatibility when the extracellular matrix (ECM) is collagen-based. Regarding the mechanical properties (MP), great efforts in tissue engineering are focused in tailoring TE properties by controlling ECM composition and organization. When cells are seeded, the collagen network is remodeled by cell-driven compaction and consolidation, produced mainly through the mechanical stimuli that can be directed selecting the geometry and the surfaces exposed to the cells. Collagen gels have different (chemical and mechanical) properties depending on their origin and preparation conditions. The MP of the collagen network are derived from the degree of cross-linking (CLD) which can be modified by different treatments. One of the techniques to evaluate MP in the network is by ultrasound (US). In this work we analyse the effect of several mechanical constraints (similar to that imposed to promote cell growth on certain sample surfaces, when seeded) on samples of gelatin with a specific geometry (thick walls cylinders) under loading conditions of pulsatile flow. We checked US parameters and estimates evolution of the network structure for different restrictions in the sample mobility. It was implemented by adapting devices specially built to measure elastic properties of biological tissues by US. The material (origin and purity) and the preparation conditions for the gelatin were selected in order to compare the results with those of literature.

An analysis of the pharmacokinetic parameter ratios in DCE-MRI using the reference region model

Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is performed by obtaining sequential MRI images, before, during and after the injection of a contrast agent. It is usually used to observe the exchange of contrast agent between the vascular space and extravascular extracellular space (EES), and provide information about blood volume and microvascular permeability. To estimate the kinetic parameters derived from the pharmacokinetic model, accurate knowledge of the arterial input function (AIF) is very important. However, the AIF is usually unknown, and it remains very difficult to obtain such information noninvasively. In this article, without knowledge of the AIF, we applied a reference region (RR) model to analyze the kinetic parameters. The RR model usually depends on kinetic parameters found in previous studies of a reference region. However, both the assignment of reference region parameters (intersubject variation) and the selection of the reference region itself (intrasubject variation) may confound the results obtained by RR methods. Instead of using literature values for those pharmacokinetic parameters of the reference region, we proposed to use two pharmacokinetic parameter ratios between the tissue of interest (TOI) and the reference region. Specifically, one parameter K R is calculated as the ratio between the volume transfer constant K trans of the TOI and RR. Similarly, another parameter V R is calculated as the ratio between the extravascular extracellular volume fraction v e of the TOI and RR. To investigate the consistency of the two ratios, the K trans of the RR was varied ranging from 0.1 to 1.0 min −1, covering the cited literature values. A simulated dataset with different levels of Gaussian noises and an in vivo dataset acquired from five canine brains with spontaneous occurring brain tumors were used to study the proposed ratios. It is shown from both datasets that these ratios are independent of K trans of the RR, implying that there is potentially no need to obtain information about literature values from the reference region for future pharmacokinetic modeling and analysis.