Sensitivity and specificity of the empirical lymphocyte genome sensitivity (LGS) assay: implications for improving cancer diagnostics.

Lymphocyte responses from 208 individuals: 20 with melanoma, 34 with colon cancer, and 4 with lung cancer (58), 18 with suspected melanoma, 28 with polyposis, and 10 with COPD (56), and 94 healthy volunteers were examined. The natural logarithm of the Olive tail moment (OTM) was plotted for exposure to UVA through 5 different agar depths (100 cell measurements/depth) and analyzed using a repeated measures regression model. Responses of patients with cancer plateaued after treatment with different UVA intensities, but returned toward control values for healthy volunteers. For precancerous conditions and suspected cancers, intermediate responses occurred. ROC analysis of mean log OTMs, for cancers plus precancerous/suspect conditions vs. controls, cancer vs. precancerous/suspect conditions plus controls, and cancer vs. controls, gave areas under the curve of 0.87, 0.89, and 0.93, respectively (P<0.001). Optimization allowed test sensitivity or specificity to approach 100% with acceptable complementary measures. This modified comet assay could represent a stand-alone test or an adjunct to other investigative procedures for detecting cancer.

Effects of different transforming growth factor beta (TGF-ß) isomers on wound closure of bone cell monolayers.

This study aimed at determining the role of the transforming growth factor-beta (TGF-ß) isomers and their combinations in bone cell behaviour using MG63 cells. The work examined how TGF-ß1, 2 and 3 and their solvent and carrier (HCl and BSA, respectively) effected cell morphology, cell proliferation and integrin expression. This study also aimed at examining how the TGF-ßs and their solvent and carrier influenced wound closure in an in vitro wound closure model and how TGF-ßs influence extracellular matrix (ECM) secretion and integrin expression. The wound healing response in terms of healing rate to the TGF-ßs and their solvent/carrier was investigated in 300 µm ± 10-30 µm SD wide model wounds induced in fully confluent monolayers of MG63 bone cells. The effect of different TGF-ß isomers and their combinations on proliferation rate and cell length of human bone cells were also assessed. Immunostaining was used to determine if TGF-ßs modifies integrin expression and ECM secretion by the bone cells. Imaging with WSPR allowed observation of the focal contacts without the need for immunostaining. The wound healing results indicated that TGF-ß3 has a significant effect on the wound healing process and its healing rate was found to be higher than the control (p < 0.001), TGF-ß1 (p < 0.001), TGF-ß2 (p < 0.001), BSA/HCl (p < 0.001) and HCl (p < 0.001) in ascending order. It was also found that TGF-ß1 and TGF-ß2 treatment significantly improved wound closure rate in comparison to the controls (p < 0.001). All TGF-ß combinations induced a faster healing rate than the control (p < 0.001). It was expected that the healing rate following treatment with TGF-ß combinations would be greater than those healing rates following treatments with TGF-ß isomers alone, but this was not the case. The results also suggest that cell morphological changes were observed significantly more in cells treated with TGF-ß(2 + 3) and TGF-ß(1 + 3) (p < 0.001). Any cell treated with TGF-ß1, TGF-ß(1 + 2) and TGF-ß(1 + 2 + 3) showed significantly less elongation compared to the control and other TGF-ß isomers. In terms of proliferation rate, TGF-ß3 and TGF-ß(2 + 3) increased cell numbers more than TGF-ß1, TGF-ß2 and other combinations. TGF-ß1 and its combinations did not show significant proliferation and attachment compared to the control. Immunostaining indicated that treatment with TGF-ß3 significantly enhanced the secretion of collagen type I, fibronectin and integrins α3 and ß1. The WSPR experiments also indicated that TGF-ßs influenced the distribution of focal contacts. In conclusion, combining TGF-ß3 with any other TGF-ß isomer resulted in a faster model wound closure rate (p < 0.001), while treatment with TGF-ß1 in any TGF-ß combination reduced the healing rate (p < 0.001). It can therefore be concluded that the presence of TGF-ß1 has an inhibitory effect on bone wound healing while TGF-ß3 had the opposite effect and increased the rate of wound closure in a 2 dimensional cell culture environment.

Microengineered surface topography facilitates cell grafting from a prototype hydrogel wound dressing with antibacterial capability.

Skin wounds derive therapeutic benefit from redeployment of dermal tissues, whether as split-thickness allo- and autografts or as biological dressings comprising cultured cells. However, the clinical outcome is strongly influenced by the techniques used for cell/tissue grafting and also the microbiological status of the wound. Here we report that microtopography incorporated into the surface of a novel polymeric material, derivatized with fibronectin to promote attachment and encourage motility, improved the efficiency of cell transfer onto de-epithelialized human skin ex vivo. The microtopography had two functions, first as a conduit for migrating cells to cross between the vehicle and recipient surface and second to shield adherent cells from destruction by mechanical shearing during handling and application. Quantitative analysis showed that topographic projections (columns) rather than recesses (pits) in the hydrogel surface achieved the highest efficiency of cell transfer. In order to address the crucial relevance of microbiological contamination to the success of wound grafting, the effect of iodine on several common bacterial pathogens was examined using an XTT+C(Q10) kinetic cell viability assay. Increasing concentrations of iodine initially stressed and after 0.5% v/v were subsequently bacteriocidal for Gram-negative Pseudomonas aeruginosa and Escherichia coli and Gram-positive Bacillus subtillis and Staphylococcus aureus. Slightly higher doses of iodine (approx 1-1.5% v/v) were required to kill HaCaT cells outright, but for both pro- and eukaryotes the major determinant of cytotoxicity was absolute dose rather than duration of exposure. Iodine delivered by the hydrogel at low concentration was bacteriostatic but not apparently cytotoxic to epithelial cells as measured by MTT end-point cell viability assay. Zone of inhibition studies confirmed that bacteriocidal quantities of neomycin, phenol red, and silver could also be delivered using the same hydrogel. This research suggests that grafting cell-based biological dressings to wounds using a topographically modified hydrogel dressing capable of simultaneous reducing the microbiological threat to a successful outcome may be a realistic clinical proposition.

Tracking traction force changes of single cells on the liquid crystal surface.

Cell migration is a key contributor to wound repair. This study presents findings indicating that the liquid crystal based cell traction force transducer (LCTFT) system can be used in conjunction with a bespoke cell traction force mapping (CTFM) software to monitor cell/surface traction forces from quiescent state in real time. In this study, time-lapse photo microscopy allowed cell induced deformations in liquid crystal coated substrates to be monitored and analyzed. The results indicated that the system could be used to monitor the generation of cell/surface forces in an initially quiescent cell, as it migrated over the culture substrate, via multiple points of contact between the cell and the surface. Future application of this system is the real-time assaying of the pharmacological effects of cytokines on the mechanics of cell migration.

Transforming growth factor beta (TGF-ß) isomers influence cell detachment of MG-63 bone cells.

Bone repair and wound healing are modulated by different stimuli. There is evidence that Transforming Growth Factor-beta (TGF-ß) super-family of cytokines have significant effects on bone structure by regulating the replication and differentiation of chondrocytes, osteoblasts and osteoclasts. There is also significant evidence that interactions with extracellular matrix molecules influence cell behaviour. In this study cell surface attachment was examined via a trypsinization assay using various TGF-ß isomers in which the time taken to trypsinize cells from the surface provided a means of assessing the strength of attachment. Three TGF-ß isomers (TGF-ß1, 2 and 3), four combined forms (TGF-ß(1+2), TGF-ß(1+3), TGF-ß(2+3) and TGF-ß(1+2+3)) along with four different controls (BSA, HCl, BSA/HCl and negative control) were investigated in this study. The results indicated that treatment with TGF-ß1, 2, 3 and HCl decreased cell attachment, however, this effect was significantly greater in the case of TGF-ß3 (p<0.001) indicating perhaps that TGF-ß3 does not act alone in cell detachment, but instead functions synergistically with signalling pathways that are dependent on the availability of hydrogen ions. Widefield Surface Plasmon Resonance (WSPR) microscope was also used to investigate cell surface interactions.

Biological imaging with a near-field optical setup.

Noncontact scanning near-field optical microscope (SNOM) systems can be used to optically resolve samples in atmospheric conditions at theoretical resolutions comparable to those of transmission electron microscope and atomic force microscope systems. SNOM systems are also increasingly used to image biological samples. In this study we custom built a SNOM system with the aim of further demonstrating the potential applications of near-field optical examination of biological material. In this study we were able to image both fixed whole-cell samples in air and liquid environments and live whole-cell samples in liquids. The images acquired were of a relatively low resolution, but this work has shown that SNOM systems can be used to monitor the dynamics of living cells at subnanometric resolutions in the z axis and for fluorescent imaging of whole cells in a liquid medium.

Biophysical characteristics of cells cultured on cholesteryl ester liquid crystals.

This study aimed at examining the biophysical characteristics of human derived keratinocytes (HaCaT) cultured on cholesteryl ester liquid crystals (CELC). CELC was previously shown to improve sensitivity in sensing cell contractions. Characteristics of the cell integrin expressions and presence of extracellular matrix (ECM) proteins on the liquid crystals were interrogated using various immunocytochemical techniques. The investigation was followed by characterization of the chemical properties of the liquid crystals (LC) after immersion in cell culture media using Fourier transform infrared spectroscopy (FTIR). The surface morphology of cells adhered to the LC was studied using atomic force microscopy (AFM). Consistent with the expressions of the integrins α2, α3 and ß1, extracellular matrix proteins (laminin, collagen type IV and fibronectin) were found secreted by the HaCaT onto CELC and these proteins were also secreted by cells cultured on the glass substrates. FTIR analysis of the LC revealed the existence of spectrum assigned to cholesterol and ester moieties that are essential compounds for the metabolizing activities of keratinocytes. The immunostainings indicated that cell adhesion on the LC is mediated by self-secreted ECM proteins. As revealed by the AFM imaging, the constraint in cell membrane spread on the LC leads to the increase in cell surface roughness and thickness of cell membrane. The biophysical expressions of cells on biocompatible CELC suggested that CELC could be a new class of biological relevant material.

Interfacial study of cell adhesion to liquid crystals using widefield surface plasmon resonance microscopy.

Widefield surface plasmon resonance (WSPR) microscopy provides high resolution imaging of interfacial interactions. We report the application of the WSPR imaging system in the study of the interaction between keratinocytes and liquid crystals (LC). Imaging of fixed keratinocytes cultured on gold coated surface plasmon substrates functionalized with a thin film of liquid crystals was performed in air using a 1.45NA objective based system. Focal adhesion of the cells adhered to glass and LC were further studied using immunofluorescence staining of the vinculin. The imaging system was also simulated with 2×2 scattering matrix to investigate the optical reflection of the resonant plasmonic wave via the glass/gold/cell and glass/gold/LC/cell layers. WSPR imaging indicated that keratinocytes are less spread and formed distinct topography of cell-liquid crystal couplings when cultured on liquid crystal coated substrates. The simulation indicates that glass/LC shifted the surface plasmon excitation angle to 75.39° as compared to glass/air interface at 44°. The WSPR microcopy reveals that the cells remodelled their topography of adhesion at different interfaces.

Contact guidance in human dermal fibroblasts is modulated by population pressure.

Morphogenesis is underpinned by orientated cell division, motility and growth. The substratum for migrating cells in vivo comprises either extracellular matrix or the surfaces of adjacent cells and both are believed to inform the dynamic behaviour of adherent cells through contact guidance. Collisions between migrating cells in vitro can induce the phenomena of contact inhibition of locomotion and division, suggesting that their sensitivity to substratum-derived cues may also be influenced by population density. In the present study dermal fibroblasts, which are known to be motile in culture and are fundamental to the organization of the extracellular matrix, were used to examine the influence of population pressure on the ability of substratum topography to induce contact guidance. The findings suggest that sensitivity to substratum-derived morphogenetic guidance cues, as revealed by alignment of cells to microtopography, is modulated by population pressure.

Motogenic substrata and chemokinetic growth factors for human skin cells.

Extracellular matrix remodelling and accurate spatio-temporal coordination of growth factor expression are two factors that are believed to regulate mitoses and cell migration in developing and regenerating tissues. The present quantitative videomicroscopical study examined the influence of some of the principal components of extracellular matrix and several growth factors that are known to be expressed in dermal wounds on three important facets of human skin cell behaviour in culture. Keratinocytes, melanocytes and dermal fibroblasts (and myofibroblast controls) exhibited varying degrees of substrate adhesion, division and migration depending on the composition of the culture substrate. Substrates that are recognized components of transitional matrices generally accentuated cell adhesion and proliferation, and were motogenic, when compared with serum-treated control surfaces, whereas components of more stable structures such as basement membrane had less influence. Platelet-derived growth factor (PDGF), epidermal growth factor (EGF) and alpha fibroblastic growth factor (alphaFGF) all promoted cell proliferation and were chemokinetic to dermal fibroblasts, but not keratinocyte growth factor (KGF) or transforming growth factor beta (TGFbeta). PDGF, EGF and KGF, but not TGFbeta or alphaFGF, all enhanced proliferation of dermal keratinocytes. The same growth factors, and in addition KGF, all stimulated motility in keratinocytes, but TGFbeta and alphaFGF again had no effect. Developing a better understanding of the interdependency of factors that control crucial cell behaviour may assist those who are interested in the regulation of histogenesis and also inform the development of rational therapeutic strategies for the management of chronic and poorly healed wounds.