A potent nonporphyrin class of photodynamic therapeutic agent: Cellular localisation, cytotoxic potential and influence of hypoxia

We have developed a totally new class of nonporphyrin photodynamic therapeutic agents with a specific focus on two lead candidates azadipyrromethene (ADPM)01 and ADPM06. Confocal laser scanning microscopy imaging showed that these compounds are exclusively localised to the cytosolic compartment, with specific accumulation in the endoplasmic reticulum and to a lesser extent in the mitochondria. Light-induced toxicity assays, carried out over a broad range of human tumour cell lines, displayed EC50 values in the micro-molar range for ADPM01 and nano-molar range for ADPM06, with no discernable activity bias for a specific cell type. Strikingly, the more active agent, ADPM06, even retained significant activity under hypoxic conditions. Both photosensitisers showed low to nondeterminable dark toxicity. Flow cytometric analysis revealed that ADPM01 and ADPM06 were highly effective at inducing apoptosis as a mode of cell death. The photophysical and biological characteristics of these PDT agents suggest that they have potential for the development of new anticancer therapeutics. © 2005 Cancer Research UK.

Mineralization capacity of Runx2/Cbfa1-genetically engineered fibroblasts is scaffold dependent

Development of tissue-engineered constructs for skeletal regeneration of large critical-sized defects requires the identification of a sustained mineralizing cell source and careful optimization of scaffold architecture and surface properties. We have recently reported that Runx2-genetically engineered primary dermal fibroblasts express a mineralizing phenotype in monolayer culture, highlighting their potential as an autologous osteoblastic cell source which can be easily obtained in large quantities. The objective of the present study was to evaluate the osteogenic potential of Runx2-expressing fibroblasts when cultured in vitro on three commercially available scaffolds with divergent properties: fused deposition-modeled polycaprolactone (PCL), gas-foamed polylactide-co-glycolide (PLGA), and fibrous collagen disks. We demonstrate that the mineralization capacity of Runx2-engineered fibroblasts is scaffold dependent, with collagen foams exhibiting ten-fold higher mineral volume compared to PCL and PLGA matrices. Constructs were differentially colonized by genetically modified fibroblasts, but scaffold-directed changes in DNA content did not correlate with trends in mineral deposition. Sustained expression of Runx2 upregulated osteoblastic gene expression relative to unmodified control cells, and the magnitude of this expression was modulated by scaffold properties. Histological analyses revealed that matrix mineralization co-localized with cellular distribution, which was confined to the periphery of fibrous collagen and PLGA sponges and around the circumference of PCL microfilaments. Finally, FTIR spectroscopy verified that mineral deposits within all Runx2-engineered scaffolds displayed the chemical signature characteristic of carbonate-containing, poorly crystalline hydroxyapatite. These results highlight the important effect of scaffold properties on the capacity of Runx2-expressing primary dermal fibroblasts to differentiate into a mineralizing osteoblastic phenotype for bone tissue engineering applications.

Osteogenic induction of human bone marrow-derived mesenchymal progenitor cells in novel synthetic polymer-hydrogel matrices

The aim of this project was to investigate the in vitro osteogenic potential of human mesenchymal progenitor cells in novel matrix architectures built by means of a three-dimensional bioresorbable synthetic framework in combination with a hydrogel. Human mesenchymal progenitor cells (hMPCs) were isolated from a human bone marrow aspirate by gradient centrifugation. Before in vitro engineering of scaffold-hMPC constructs, the adipogenic and osteogenic differentiation potential was demonstrated by staining of neutral lipids and induction of bone-specific proteins, respectively. After expansion in monolayer cultures, the cells were enzymatically detached and then seeded in combination with a hydrogel into polycaprolactone (PCL) and polycaprolactone-hydroxyapatite (PCL-HA) frameworks. This scaffold design concept is characterized by novel matrix architecture, good mechanical properties, and slow degradation kinetics of the framework and a biomimetic milieu for cell delivery and proliferation. To induce osteogenic differentiation, the specimens were cultured in an osteogenic cell culture medium and were maintained in vitro for 6 weeks. Cellular distribution and viability within three-dimensional hMPC bone grafts were documented by scanning electron microscopy, cell metabolism assays, and confocal laser microscopy. Secretion of the osteogenic marker molecules type I procollagen and osteocalcin was analyzed by semiquantitative immunocytochemistry assays. Alkaline phosphatase activity was visualized by p-nitrophenyl phosphate substrate reaction. During osteogenic stimulation, hMPCs proliferated toward and onto the PCL and PCL-HA scaffold surfaces and metabolic activity increased, reaching a plateau by day 15. The temporal pattern of bone-related marker molecules produced by in vitro tissue-engineered scaffold-cell constructs revealed that hMPCs differentiated better within the biomimetic matrix architecture along the osteogenic lineage.

The role of inflammation in the pathogenesis of non-small cell lung cancer

The link between chronic immune activation and tumorigenesis is well established. Compelling evidence has accumulated that histologic assessment of infiltration patterns of different host immune response components in non-small cell lung cancer specimens helps identify different prognostic patient subgroups. This review provides an overview of recent insights gained in the understanding of the role played by chronic inflammation in lung carcinogenesis. The usefulness of quantification of different populations of lymphocytes, natural killer cells, macrophages, and mast cells within the tumor microenvironment in non-small cell lung cancer is also discussed. In particular, the importance of assessment of inflammatory cell microlocalization within both the tumor islet and surrounding stromal components is emphasized. Copyright © 2010 by the International Association for the Study of Lung Cancer.

Carbonic anhydrase IX expression, a novel surrogate marker of tumor hypoxia, is associated with a poor prognosis in non-small-cell lung cancer

Purpose To evaluate carbonic anhydrase (CA) IX as a surrogate marker of hypoxia and investigate the prognostic significance of different patterns of expression in non-small-cell lung cancer (NSCLC). Methods Standard immunohistochemical techniques were used to study CA IX expression in 175 resected NSCLC tumors. CA IX expression was determined by Western blotting in A549 cell lines grown under normoxic and hypoxic conditions. Measurements from microvessels to CA IX positivity were obtained. Results CA IX immunostaining was detected in 81.8% of patients. Membranous (m) (P = .005), cytoplasmic (c) (P = .018), and stromal (P < .001) CA IX expression correlated with the extent of tumor necrosis (TN). The mean distance from vascular endothelium to the start of tumor cell positivity was 90 μm, which equates to an oxygen pressure of 5.77 mmHg. The distance to blood vessels from individual tumor cells or tumor cell clusters was greater if they expressed mCA IX than if they did not (P < .001). Hypoxic exposure of A549 cells for 16 hours enhanced CAIX expression in the nuclear and cytosolic extracts. Perinuclear (p) CA IX (P = .035) was associated with a poor prognosis. In multivariate analysis, pCA IX (P = .004), stage (P = .001), platelet count (P = .011), sex (P = .027), and TN (P = .035) were independent poor prognostic factors. Conclusion These results add weight to the contention that mCA IX is a marker of tumor cell hypoxia. The absence of CA IX staining close to microvessels suggests that these vessels are functionally active. pCA IX expression is representative of an aggressive phenotype. © 2003 by American Society of Clinical Oncology.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 μM, and inhibition is complete at about 10 μM. In this range, the tubulin assembly is fully (up to 6 μM) or partially (∼6-10 μM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect- concentration for inhibition of microtubule assembly in vitro was 1 μM Hg2+, the IC50 5.8 μM. Mercury(II) salts at the IC 50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a "gliding assay" mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 μM and a complete inhibition is reached at 1 μM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 μM HgCl2. Between 15 and 20 μM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 μM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations (100 μM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg 2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Memory and Epileptogenesis in Complex Biological and Simulated Systems

Oscillations of neural activity may bind widespread cortical areas into a neural representation that encodes disparate aspects of an event. In order to test this theory we have turned to data collected from complex partial epilepsy (CPE) patients with chronically implanted depth electrodes. Data from regions critical to word and face information processing was analyzed using spectral coherence measurements. Similar analyses of intracranial EEG (iEEG) during seizure episodes display HippoCampal Formation (HCF)—NeoCortical (NC) spectral coherence patterns that are characteristic of specific seizure stages (Klopp et al. 1996). We are now building a computational memory model to examine whether spatio-temporal patterns of human iEEG spectral coherence emerge in a computer simulation of HCF cellular distribution, membrane physiology and synaptic connectivity. Once the model is reasonably scaled it will be used as a tool to explore neural parameters that are critical to memory formation and epileptogenesis.

Diverse populations of T cells with NK cell receptors accumulate in the human intestine in health and in colorectal cancer

T cells expressing NK cell receptors (NKR) display rapid MHC-unrestricted cytotoxicity and potent cytokine secretion and are thought to play roles in immunity against tumors. We have quantified and characterized NKR+ T cells freshly isolated from epithelial and lamina propria layers of duodenum and colon from 16 individuals with no evidence of gastrointestinal disease and from tumor and uninvolved tissue from 19 patients with colorectal cancer. NKR+ T cell subpopulations were differentially distributed in different intestinal compartments, and CD161+ T cells accounted for over one half of T cells at all locations tested. Most intestinal CD161+ T cells expressed alpha beta TCR and either CD4 or CD8. Significant proportions expressed HLA-DR,CD69 and Fas ligand. Upon stimulation in vitro, CD161+ T cells produced IFN-gamma and TNF-alpha but not IL-4. NKT cells expressing the Valpha24Vbeta11 TCR, which recognizes CD1d,were virtually absent from the intestine, but colonic cells produced IFN-gamma in response to the NKT cell agonist ligand alpha-galactosylceramide. NKR+ T cells were not expanded in colonic tumors compared to adjacent uninvolved tissue. The predominance, heterogeneity and differential distribution of NKR+ T cells at different intestinal locations suggests that they are central to intestinal immunity.

Perfusion model system to generate engineered grafts with controlled cellular distributions

Engineered tissue grafts, which mimic the spatial variations of cell density and extracellular matrix present in native tissues, could facilitate more efficient tissue regeneration and integration. We previously demonstrated that cells could be uniformly seeded throughout a 3D scaffold having a random pore architecture using a perfusion bioreactor2. In this work, we aimed to generate 3D constructs with defined cell distributions based on rapid prototyped scaffolds manufactured with a controlled gradient in porosity. Computational models were developed to assess the influence of fluid flow, associated with pore architecture and perfusion regime, on the resulting cell distribution.

Expression and distribution of cell-surface proteoglycans in the normal Lewis rat molar periodontium

Cell-surface proteoglycans participate in several biological functions such as cell cell and cell-matrix interactions, cell adhesion, the binding to various growth factors as co-receptors and repair. To understand better the expression and distribution of cell-surface proteoglycans in the periodontal tissues, an immunohistochemical evaluation of the normal Lewis rat molar periodontium using panels of antibodies for syndecan-1, -2, -4, glypican and betaglycan was carried out. Our results demonstrated the expression and distribution of all proteoglycans in the suprabasal gingival epithelium, soft and hard connective tissues. Both cellular and matrix localization was evident within the various periodontal compartments. The presence of these cell-surface proteoglycans indicates the potential for roles in the process of tissue homeostasis, repair or regeneration in periodontium of which each function requires further study.

Improved sub-cellular resolution via simultaneous analysis of organelle proteomics data across varied experimental conditions

Spatial organisation of proteins according to their function plays an important role in the specificity of their molecular interactions. Emerging proteomics methods seek to assign proteins to sub-cellular locations by partial separation of organelles and computational analysis of protein abundance distributions among partially separated fractions. Such methods permit simultaneous analysis of unpurified organelles and promise proteome-wide localisation in scenarios wherein perturbation may prompt dynamic re-distribution. Resolving organelles that display similar behavior during a protocol designed to provide partial enrichment represents a possible shortcoming. We employ the Localisation of Organelle Proteins by Isotope Tagging (LOPIT) organelle proteomics platform to demonstrate that combining information from distinct separations of the same material can improve organelle resolution and assignment of proteins to sub-cellular locations. Two previously published experiments, whose distinct gradients are alone unable to fully resolve six known protein-organelle groupings, are subjected to a rigorous analysis to assess protein-organelle association via a contemporary pattern recognition algorithm. Upon straightforward combination of single-gradient data, we observe significant improvement in protein-organelle association via both a non-linear support vector machine algorithm and partial least-squares discriminant analysis. The outcome yields suggestions for further improvements to present organelle proteomics platforms, and a robust analytical methodology via which to associate proteins with sub-cellular organelles.

The dynamics of cellular automata on 2-manifolds is affected by topology

In this paper, we demonstrate that the distribution of Wolfram classes within a cellular automata rule space in the triangular tessellation is not consistent across different topological general. Using a statistical mechanics approach, cellular automata dynamical classes were approximated for cellular automata defined on genus-0, genus-1 and genus-2 2-manifolds. A distribution-free equality test for empirical distributions was applied to identify cases in which Wolfram classes were distributed differently across topologies. This result implies that global structure and local dynamics contribute to the long term evolution of cellular automata.

On the effect of topology on cellular automata rule spaces

This thesis presents an empirical study of the effects of topology on cellular automata rule spaces. The classical definition of a cellular automaton is restricted to that of a regular lattice, often with periodic boundary conditions. This definition is extended to allow for arbitrary topologies. The dynamics of cellular automata within the triangular tessellation were analysed when transformed to 2-manifolds of topological genus 0, genus 1 and genus 2. Cellular automata dynamics were analysed from a statistical mechanics perspective. The sample sizes required to obtain accurate entropy calculations were determined by an entropy error analysis which observed the error in the computed entropy against increasing sample sizes. Each cellular automata rule space was sampled repeatedly and the selected cellular automata were simulated over many thousands of trials for each topology. This resulted in an entropy distribution for each rule space. The computed entropy distributions are indicative of the cellular automata dynamical class distribution. Through the comparison of these dynamical class distributions using the E-statistic, it was identified that such topological changes cause these distributions to alter. This is a significant result which implies that both global structure and local dynamics play a important role in defining long term behaviour of cellular automata.

Evaluation of the spatial distribution of γH2AX following ionizing radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as γH2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of γH2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of γH2AX as molecular marker of DSBs, a disparity in γ-irradiation-induced γH2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of γH2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between γH2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of γH2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3Dmodeling.