Barbara Safiejko-Mroczka

Long-term cultivation of HepG2 liver cells encapsulated in alginate hydrogels: a study of cell viability, morphology and drug metabolism.

In this study, we have evaluated the use of ultra-sterile alginate hydrogels encapsulated with HepG2 liver cells for applications in high throughput drug screening. We have studied the cellular viability and metabolic capacity of the encapsulated cells in two different alginate structures SLM100 (G:M::40:60) and SLG100 (G:M::60:40). We have also developed protocols to characterize the encapsulated cells within the alginate structure using scanning electron microscopy (SEM) and laser scanning confocal microscopy (LSCM). Further we have studied the Phase-I/II metabolic characteristics of the encapsulated cells in monolayer and 3D culture. Our results indicate that cells encapsulated within SLM100 and SLG100 class of alginates have shown high cellular viability with >80% even after 14 days in culture. As expected, the proliferation rates of the encapsulated cells are held steady and do not proliferate within the gels. Production of liver-specific enzymes such as CYP1A1 and CYP3A4 after 14 days in culture indicates the viability and functionality of the encapsulated HepG2 cells. Phase-II Glutathione activity of the encapsulated cells were also maintained in 3D culture conditions. The encapsulated cells within the 3D gels were also capable of metabolizing the pro-drug EFC (7-ethoxy-4-trifluoromethyl coumarin) to HFC (7-hydroxy-4-trifluoromethyl) in a linear fashion over a period of time. These results have provided us with baseline results to benchmark future improvements in material and design configurations for optimal pharmacokinetic response of in vitro tissue model systems.

Clostridium difficile toxin B activates dual caspase‐dependent and caspase‐independent apoptosis in intoxicated cells

Clostridium difficile toxin B (TcdB) inactivates the small GTPases Rho, Rac and Cdc42 during intoxi‐cation of mammalian cells. In the current work, we show that TcdB has the potential to stimulate caspase‐dependent and caspase‐independent apoptosis. The apoptotic pathways became evident when caspase‐3‐processed‐vimentin was detected in TcdB‐treated HeLa cells. Caspase‐3 activation was subsequently confirmed in TcdB‐intoxicated HeLa cells. Interestingly, caspase inhibitor delayed TcdB‐induced cell death, but did not alter the time‐course of cytopathic effects. A similar effect was also observed in MCF‐7 cells, which are deficient in caspase‐3 activity. The time‐course to cell death was almost identical between cells treated with TcdB plus caspase inhibitor and cells intoxicated with the TcdB enzymatic domain (TcdB1–556). Unlike TcdB treated cells, intoxication with TcdB1–556 or expression of TcdB1–556 in a transfected cell line, did not stimulate caspase‐3 activation yet cells exhibited cytopathic effects and cell death. Although TcdB1–556 treated cells did not demonstrate caspase‐3 activation these cells were apoptotic as determined by differential annexin‐V/propidium iodide staining and nucleosomal DNA fragmentation. These data indicate TcdB triggers caspase‐independent apoptosis as a result of substrate inactivation and may evoke caspase‐dependent apoptosis due to a second, yet undefined, activity of TcdB. This is the first example of a bacterial virulence factor with the potential to stimulate multiple apoptotic pathways in host cells.

Evolutionary Selection of New Breast Cancer Cell-Targeting Peptides and Phages with the Cell-Targeting Peptides Fully Displayed on the Major Coat and Their Effects on Actin Dynamics during Cell Internalization

Filamentous phage as a bacteria-specific virus can be conjugated with an anticancer drug and has been proposed to serve as a carrier to deliver drugs to cancer cells for targeted therapy. However, how cell-targeting filamentous phage alone affects cancer cell biology is unclear. Phage libraries provide an inexhaustible reservoir of new ligands against tumor cells and tissues that have potential therapeutic and diagnostic applications in cancer treatment. Some of these identified ligands might stimulate various cell responses. Here we identified new cell internalizing peptides (and the phages with such peptides fused to each of ~3900 copies of their major coat protein) using landscape phage libraries and for the first time investigated the actin dynamics when selected phages are internalized into the SKBR-3 breast cancer cells. Our results show that phages harboring VSSTQDFP and DGSIPWST peptides could selectively internalize into the SKBR-3 breast cancer cells with high affinity, and also show rapid involvement of membrane ruffling and rearrangements of actin cytoskeleton during the phage entry. The actin dynamics was studied by using live cell and fluorescence imaging. The cell-targeting phages were found to enter breast cancer cells through energy dependent mechanism and phage entry interferes with actin dynamics, resulting in reorganization of actin filaments and increased membrane rufflings in SKBR-3 cells. These results suggest that, when phage enters epithelial cells, it triggers transient changes in the host cell actin cytoskeleton. This study also shows that using multivalent phage libraries considerably increases the repertoire of available cell-internalizing ligands with potential applications in targeted drug delivery, imaging, molecular monitoring and profiling of breast cancer cells.

The relationship between receptor for advanced glycation end products expression and the severity of periodontal disease in the gingiva of diabetic and non diabetic periodontitis patients.

OBJECTIVE Activation of receptor for advanced glycation end products (RAGE) has been implicated in many chronic diseases, including diabetic complications. In this study we examined the relationship between RAGE expression and the morphological changes seen in the gingiva of diabetic and periodontitis patients. DESIGN Gingival biopsies from 15 diabetic patients with periodontitis, 25 non diabetic patients with periodontitis and 10 healthy individuals were collected. Sections were stained with haematoxylin and eosin, and immunohistochemically to detect RAGE. Samples were examined in light and fluorescence microscopes and histomorphometric analysis was performed. RESULTS Increased number of inflammatory cells and changes in collagen and vasculature were observed in diabetic and non diabetic patients with periodontitis. RAGE was weakly expressed in healthy gingiva. The strongest reaction with anti-RAGE antibody was found in the gingiva of diabetic patients with periodontitis followed by the severe periodontitis patients. RAGE expression in inflammatory cells was stronger than in the epithelium. The number of inflammatory cells in the gingiva was higher in the diabetic periodontitis patients than in the non diabetic severe periodontitis patients. CONCLUSIONS RAGE is strongly expressed in the gingiva of diabetic patients with periodontitis and with severe periodontitis alone, the latter indicating RAGE activation even in the absence of hyperglycemia. However our findings are based on relatively small sample size. With a larger patient population, some of our other findings may have reached statistical significance.

PREPARING WHOLE MOUNTS OF BIOLOGICAL SPECIMENS FOR IMAGING MACROMOLECULAR STRUCTURES BY LIGHT AND ELECTRON MICROSCOPY

In this article we critically review the procedures involved in preparing whole mounts of biological samples for microscopic observation, particularly at high resolution. We discuss practical methods for optimizing specimen preservation to achieve the two principal goals of biological specimen preparation: (a) preserving biological structures as close to their living configuration as possible, and (b) rendering them visible with the desired imaging method. Drawing on examples taken from our own work, and using fluorescence light microscopy and scanning electron microscopy to study the cytoskeleton of vertebrate cells in culture, we show that obtaining optimal results is often a compromise between maximum preservation and the clear visualization of structures. We have found that fixing cells with bifunctional protein crosslinking reagents, in combination with detergent extraction, allows internal cell structures to be preserved, labeled with specific probes, and visualized with microscopic methods. We also review and discuss the relative merits of different procedures for fixing (chemical fixation and cryofixation), drying (air‐drying, critical point‐drying, and freeze‐drying) and coating biological specimens with metals to facilitate visualization in the electron microscope.

The induction of protrusion by neomycin in human glioma cells is correlated with a decrease followed by an increase in filamentous actin.

In this paper we describe an experimental investigation of the mechanism of motility of vertebrate cells. Human glioma cells were treated with neomycin, an inhibitor of the phosphatidylinositol cycle; and changes in cell motility and the cytoskeleton were examined by video, fluorescence, and scanning electron microscopy and by cytofluorometry. Neomycin stimulates a single protrusion of lamellipodia from the cell margin, which is correlated with an initial rapid decrease in the amount of F‐actin throughout the cell, especially at the cell edge; the fragmentation of actin filaments within the lamellipodia; and the subsequent de novo polymerization of F‐actin in a marginal band at the leading edge of lamellipodia. Changes in F‐actin are paralleled by changes in the distribution and amount of gelsolin. These results support the hypothesis that protrusion is initiated by the gelsolin‐mediated severing and subsequent depolymerization of cortical actin filaments, which weakens the cell cortex, allowing hydrostatic or gel osmotic pressure to force the cell margin to protrude. The accompanying polymerization of filaments actin at the leading edge of the protrusion may stabilize the protrusion and support its expansion.

Development of an optimized protocol for studying the interaction of filamentous bacteriophage with mammalian cells by fluorescence microscopy

Filamentous bacteriophage has been proposed as a vehicle that can carry and deliver therapeutics into mammalian cells for disease treatment, thus a protocol for imaging phage‐cell interaction is essential. Because high signal intensity is necessary to study the mechanism of interaction between filamentous bacteriophage and mammalian cells, it is important to optimize the procedure for fluorescence labeling of phage in order to understand such interaction. Here, we describe a procedure that gives intense fluorescence labeling and can show interactions between fd‐tet bacteriophage selected from phage libraries and mammalian cells (SKBR‐3 and MCF‐10A). The indirect labeling of phage with dye‐conjugated antibody and cytoskeleton associated proteins was significantly enhanced in the presence of a cross‐linking reagent called dithiobissuccinimidylpropionate (DSP) as shown by qualitative and quantitative fluorescence microscopy. The use of DSP resulted in high signal intensity in fluorescence imaging of phage‐cell complex. The DSP cross‐linker is believed to preserve soluble unbound proteins for fluorescence imaging. The interaction between the phage and mammalian cells was further confirmed by scanning electron microscopy. Microsc. Res. Tech., 2010.