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M Toner

Publications and source records attributed to M Toner.

At least 91 records · Page 5Linked to original sources
PubMed1995

Detection of Epstein-Barr virus DNA in tongue tissues from AIDS autopsies without clinical evidence of oral hairy leukoplakia.

Epstein-Barr virus (EBV) DNA was detected by in situ hybridization at 3 sites of 30 samples taken from clinically normal lateral border of tongue mucosa from 15 AIDS autopsies and in none of 20 samples from 10 controls. The first positive case showed a thin layer of parakeratosis correlated with positive signals for EBV in one area and an adjacent area without obvious parakeratosis was also positive for EBV. These findings were present on both sides of the tongue. The second case was unilaterally positive for EBV and parakeratosis was absent. The hybridization signals were localised to koilocyte-like cells in the stratum spinosum, as in oral hairy leukoplakia (OHL). These observations suggest that the in situ hybridization technique can detect very early or subclinical OHL, and supports the role of EBV in the pathogenesis of this lesion.

Acquired Immunodeficiency Syndrome↗
PubMed1994

Selective adhesion of hepatocytes on patterned surfaces.

Successful development of cell-biased bioartificial liver devices necessitates the establishment of techniques and designs for long-term, stable hepatocellular function and efficient transport of nutrients and wastes within the device. Given the relatively large cell mass that one must consider, one possible solution involves the use of micropatterning technology to sandwich hepatocytes aligned in rows between two micropatterned surfaces. Rows of cells would alternate with hepatocyte-free areas, creating efficient transport channels for fluid flow and nutrient exchange. Ultimately, this type of device could also be used as a three-dimensional construct for investigating a variety of cell-surface, cell-extracellular matrix, and cell-cell interactions. To achieve this goal, one must develop techniques for selectively adhering hepatocytes to solid substrates. In this study, reproducible, selective adhesion of hepatocytes on a glass substrate with large regions of adhesive (AS) and nonadhesive (NAS) surfaces was obtained. The AS had hydrophilic characteristics, enhancing deposition of collagen molecules from an aqueous solution, and subsequent hepatocyte adhesion, whereas the NAS had hydrophobic properties and remained collagen-free and hepatocyte-free. In addition, a reproducible processing technique for obtaining patterns of hepatocytes was developed and optimized, using a surface with a single AS band as a first approximation to a micropatterned device. This was achieved by spincoating an aqueous collagen type I solution (0.1 mg/mL) on a banded surface at 500 rpm for 25 seconds. The morphology and long-term function of the hepatocytes attached to AS in nonbanded and banded surface configurations was assessed by mimicking sandwich culture and was shown to be similar to stable, differentiated sandwich cultures. Mathematical modeling was used to determine critical design criteria for the hypothetical micropatterned device. The oxygen distribution and viscous pressure drop were modeled along a typical microchannel and limited to in vivo values. An optimal channel length of 0.6 cm and a flow rate of 2.0 x 10(-6) mL/s were obtained for a channel of 100 microns in width and 10 microns in height. These values were reasonable in terms of practical implementation.

Albumins↗
PubMed1994

The prognostic significance of immunohistochemically detected lymph node micrometastases in colorectal carcinoma.

Micrometastases have been detected by immunocytochemical means in the lymph nodes of patients with otherwise node-negative cancer of the colon and rectum. This study examines the incidence and prognostic significance of nodal micrometastases in Dukes' B carcinoma. Five hundred and fifty-nine lymph nodes from 77 cases of Dukes' B carcinoma were examined for lymph node micrometastases by immunocytochemical staining for cytokeratin AE1:AE3. Micrometastases were detected in 19 cases (25 per cent). Cell clusters were present in ten cases, the remaining nine cases displaying only single cells. The presence of micrometastases was unrelated to age (P = 0.06), sex (P = 0.32), tumour site (P = 0.37), tumour size (P = 0.67), or tumour differentiation (P = 0.66). Ten-year survival estimates by the Kaplan-Meier lifetable method was 47 per cent in patients with and without micrometastases (chi 2 = 0.35 and 1 df, P = ns). The presence of nodal micrometastases detectable only by immunocytochemistry in patients with Dukes' B colorectal cancer does not justify reassignment to a more advanced disease stage.

Aged↗
PubMed1994

Antibody-targeted photolysis of bacteria in vivo.

We have evaluated the efficacy of antibody-targeted photolysis to kill bacteria in vivo using specific antibacterial photosensitizer (PS) immunconjugates. After infecting the dorsal skin in mice with Pseudomonas aeruginosa, both specific and nonspecific tin (IV) chlorin e6-monoclonal antibody conjugates were injected at the infection site. After a 15 min incubation period, the site was exposed to 630 nm light with a power density of 100 mW/cm2 for 1600 seconds. Irradiation resulted in a greater then 75% decrease in the number of viable bacteria at sites treated with a specific conjugate, whereas normal bacterial growth was observed in animals that were untreated or treated with a nonspecific conjugate. Antibody-targeted photolysis may be a selective and versatile tool for treating a variety of infections.

Animals↗
PubMed1994

Transport of fluorescent dextrans across the rat ileum after cutaneous thermal injury.

OBJECTIVE: To determine the time course and spatial distribution of uptake of macromolecules in the small intestine of rats subjected to cutaneous thermal injury. DESIGN: Prospective, controlled animal study. SUBJECTS: Fifty-five female Sprague-Dawley (CD) rats subjected to scald burn injury covering 20% (small injury; n = 29) and 40% (large injury; n = 6) of the total body surface area between 3 and 72 hrs after injury. Animals subjected to sham injury (n = 20) were used as controls. INTERVENTIONS: The intestine was cannulated near the distal ileum and incised 7 cm upstream. After perfusion with physiologic buffer, this intestinal loop was filled with the same buffer containing fluorescent-labeled dextrans (3 and 70 kilodaltons molecular weight) and ligated 4 cm from the injection point. After a 2-hr incubation period, the tissues were fixed with paraformaldehyde and cryosections were examined by laser confocal microscopy. The mesentery was also observed by laser confocal microscopy during incubation with the permeability probes. The disappearance of fluorescence was studied after washing the dextran probes from the gut lumen. MEASUREMENTS AND MAIN RESULTS: In small injuries, there was a transient uptake of the 3-kilodalton dextran by the epithelium in focal regions of the ileum with the effects seen between 7 and 21 hrs after injury. In large injuries, epithelial staining was visible within 3 hrs, and the marker was seen to translocate both to the lymphatics and the blood vessels of the mesentery. In comparison, the 70-kilodalton dextran was visible within the intercellular spaces. Little or no epithelial staining was seen in sham-injured animals. CONCLUSIONS: These results suggest that a transcellular pathway for the translocation of small macromolecules from the lumen to the mesentery can be activated after burn injury. The novel techniques described here will be useful to examine intestinal transport in various pathologic situations.

Animals↗
PubMed1994

In situ hybridization and the polymerase chain reaction (PCR) in the analysis of biopsies and exfoliative cytology specimens for definitive diagnosis of oral hairy leukoplakia (OHL).

The definitive diagnosis of oral hairy leukoplakia (OHL) demands that Epstein-Barr virus (EBV) is demonstrated in the lesional tissue, since the histopathological features on conventional light microscopy are not pathognomonic. We have investigated the possible use of polymerase chain reaction (PCR) technology in reaching a definitive diagnosis of this lesion by its application to ten biopsy specimens with definitive diagnoses of OHL determined by in situ hybridization. EBV DNA was demonstrated by PCR in all ten OHL biopsy specimens analysed, and none of ten control specimens. Furthermore, we have investigated the role of PCR in analysis of exfoliative cytology samples collected from the lateral border of the tongue by a minimally-invasive scraping technique. EBV DNA was not only detected in all OHL lesional scrapings but also in more than one-third of healthy controls, due to viral presence in saliva at the time of sampling. In this application, the highly sensitive PCR technique has low specificity and cannot be recommended.

Adolescent↗
PubMed1994

An extracorporeal microscopy perfusion chamber for on-line studies of environmental effects on cultured hepatocytes.

The development of bioartificial-hybrid organ support systems is hampered by the lack of knowledge on the effects of different (in vivo) environments on cells during extracorporeal perfusion. In the present study, a perfusion chamber was designed for continuous monitoring of cultured cells during perfusion with media, as well as during plasma perfusion in an extracorporeal circuit. Chamber characterization showed satisfactory thermal and perfusion profiles and no major pH fluctuations. Further testing was performed with hepatocytes that were cultured in between two collagen layers, a configuration which was previously shown to preserve hepatocyte morphology and function for over six weeks of culture. Perfusion of the hepatocytes with culture media did not adversely affect cell morphology and function, provided the perfusion time was < or = 48 hours. Perfusion of the cultures during connection of the chamber to an extracorporeal circuit involving normal rats for six hours resulted in reversible cytoplasmic changes, unaltered cell shapes indices, and a 40 percent increase in albumin secretion rate during the first post-perfusion day, followed by a return to stable control levels. We expect that this chamber will be a valuable tool for on-line studies of cells under (extracorporeal) perfusion conditions and could be used for a large variety of studies on regeneration, reperfusion damage, and detoxification.

Albumins↗
PubMed1993

Effect of collagen gel configuration on the cytoskeleton in cultured rat hepatocytes.

The cytoskeleton is important in the maintenance of cellular morphology and differentiated function in a number of cell types, including hepatocytes. In this study, adult rat hepatocytes sandwiched between two layers of collagen gel were compared to cells cultured on a single collagen gel for differences in the organization and expression of the cytoskeletal proteins actin and tubulin. Hepatocytes cultured between two layers of hydrated rat tail tendon collagen (sandwich gel) morphologically resembled cells in intact liver for several weeks. Actin filaments (F-actin) in these hepatocytes were concentrated under the plasma membrane in regions of cell-cell contact. In contrast, hepatocytes cultured on a single collagen gel were flattened and motile and had F-actin containing stress fibers. This was accompanied by a severalfold increase in actin mRNA. Microtubules formed an interwoven network in hepatocytes cultured in a sandwich gel, but in single gel cultures they formed long parallel arrays extending out to the cell periphery. Tubulin mRNA was severalfold greater in hepatocytes cultured on a single gel. Fibronectin and laminin staining were greater in single gel cultures, and these proteins were concentrated in fibrils radiating from the cell periphery. Overlaying a second collagen gel onto hepatocytes that had been cultured on a single gel (double gel rescue) reversed cell spreading and reduced stress fibers. Double gel rescue also resulted in a decrease in actin and tubulin mRNA to levels present in sandwich gel cultures and freshly isolated hepatocytes. These results show that the configuration of the external matrix has a dynamic effect on cytoskeletal proteins in cultured rat hepatocytes.

Actins↗
PubMed1993

Immobilized IL-2 preserves the viability of an IL-2 dependent cell line.

The mouse T-cell line CTLL-2 is known to be dependent on interleukin-2 (IL-2) for both growth and viability. These cells possess high affinity IL-2 receptors and have been shown to internalize IL-2 after binding. To determine if internalization of IL-2 is required for the mediation of its signal, IL-2 was covalently coupled to an insoluble matrix via glutaraldehyde cross-linking and CTLL-2 cells were incubated with the immobilized lymphokine matrix. This covalent cross-linking prevents the free lateral diffusion and internalization of the bound IL-2 receptors (IL-2R) while still permitting specific binding between the cells and the immobilized ligands. Although only very limited proliferation was observed during the incubation as assessed by 3H-thymidine incorporation, the viability of the CTLL-2 cells on the immobilized IL-2 matrix was preserved. Cells incubated on the immobilized IL-2 surface could proliferate in response to exogenous soluble IL-2 that was added to the cultures after 36 hours whereas control cultures incubated with an immobilized BSA matrix had died. This indicates that immobilized IL-2 can mediate some of the activity of soluble IL-2 and that internalization of the IL-2 receptor may not be required for at least part of the IL-2 mediated effect.

Animals↗
PubMed1993

Nucleation and growth of ice crystals inside cultured hepatocytes during freezing in the presence of dimethyl sulfoxide.

A three-part, coupled model of cell dehydration, nucleation, and crystal growth was used to study intracellular ice formation (IIF) in cultured hepatocytes frozen in the presence of dimethyl sulfoxide (DMSO). Heterogeneous nucleation temperatures were predicted as a function of DMSO concentration and were in good agreement with experimental data. Simulated freezing protocols correctly predicted and explained experimentally observed effects of cooling rate, warming rate, and storage temperature on hepatocyte function. For cells cooled to -40 degrees C, no IIF occurred for cooling rates less than 10 degrees C/min. IIF did occur at faster cooling rates, and the predicted volume of intracellular ice increased with increasing cooling rate. Cells cooled at 5 degrees C/min to -80 degrees C were shown to undergo nucleation at -46.8 degrees C, with the consequence that storage temperatures above this value resulted in high viability independent of warming rate, whereas colder storage temperatures resulted in cell injury for slow warming rates. Cell damage correlated positively with predicted intracellular ice volume, and an upper limit for the critical ice content was estimated to be 3.7% of the isotonic water content. The power of the model was limited by difficulties in estimating the cytosol viscosity and membrane permeability as functions of DMSO concentration at low temperatures.

Animals↗
PubMed1993

Nonequilibrium freezing of one-cell mouse embryos. Membrane integrity and developmental potential.

A thermodynamic model was used to evaluate and optimize a rapid three-step nonequilibrium freezing protocol for one-cell mouse embryos in the absence of cryoprotectants (CPAs) that avoided lethal intracellular ice formation (IIF). Biophysical parameters of one-cell mouse embryos were determined at subzero temperatures using cryomicroscopic investigations (i.e., the water permeability of the plasma membrane, its temperature dependence, and the parameters for heterogeneous IIF). The parameters were then incorporated into the thermodynamic model, which predicted the likelihood of IIF. Model predictions showed that IIF could be prevented at a cooling rate of 120 degrees C/min when a 5-min holding period was inserted at -10 degrees C to assure cellular dehydration. This predicted freezing protocol, which avoided IIF in the absence of CPAs, was two orders of magnitude faster than conventional embryo cryopreservation cooling rates of between 0.5 and 1 degree C/min. At slow cooling rates, embryos predominantly follow the equilibrium phase diagram and do not undergo IIF, but mechanisms other than IIF (e.g., high electrolyte concentrations, mechanical effects, and others) cause cellular damage. We tested the predictions of our thermodynamic model using a programmable freezer and confirmed the theoretical predictions. The membrane integrity of one-cell mouse embryos, as assessed by fluorescein diacetate retention, was approximately 80% after freezing down to -45 degrees C by the rapid nonequilibrium protocol derived from our model. The fact that embryos could be rapidly frozen in the absence of CPAs without damage to the plasma membrane as assessed by fluorescein diacetate retention is a new and exciting finding. Further refinements of this protocol is necessary to retain the developmental competence of the embryos.

Animals↗
PubMed1993

Cellular response of mouse oocytes to freezing stress: prediction of intracellular ice formation.

Successful protocols for cryopreservation of living cells can be designed if the physicochemical conditions to preclude intracellular ice formation (IIF) can be defined. Unfortunately, all attempts to predict the probability of IIF have met with very limited success. In this study, an analytical model is developed to predict ice formation inside mouse oocytes subjected to a freezing stress. According to the model, IIF is catalyzed heterogeneously by the plasma membrane (i.e., surface catalyzed nucleation, SCN). A local site on the plasma membrane is assumed to become an ice nucleator in the presence of the extracellular ice via its effects on the membrane. This interaction is characterized by the contact angle between the plasma membrane and the ice cluster. In addition, IIF is assumed to be catalyzed at temperatures below -30 degrees C by intracellular particles distributed throughout the cell volume (i.e., volume catalyzed nucleation, VCN). In the present study, these two distinctly coupled modes of IIF, especially SCN, are applied to various experimental protocols from mouse oocytes. Excellent agreement between predictions and observations suggests that the proposed model of IIF is adequate.

Animals↗
PubMed1993

Antibody Targeted Photolysis.

The technique of Antibody Targeted Photolysis (ATPL) is reviewed from an historical perspective with a summary of the literature since the first experiments were performed in 1983. Attention is given to both the biological and photophysical properties of the various immunoconjugates that have been developed. References to critical discoveries and competing technologies in the photodynamic literature are given. Topics include: synthesis of immunoconjugates, in vitro vs. in vivo toxicity, in vivo biodistribution, immunoconjugate delivery, photosensitizer selection based on photophysical properties, light delivery for specific applications, oxygen requirements, and other physicochemical phenomena. A mathematical model of the dynamics for cell killing based upon the transport of phototoxins to the cell surface is developed. A generalized set of coupled differential equations is given, which conveniently summarizes the manifold requirements stressed earlier for successful cell killing. Solutions are then presented for an idealized set of conditions appropriate for an isolated tumor cell. Suggestions for further improvements and follow-up experiments are made that could help in the evolution of ATPL into a useful clinical therapy and/or probe for cell biological studies.

Animals↗
PubMed1992

Effects of dimethyl sulfoxide on cultured rat hepatocytes in sandwich configuration.

A recently developed sandwich culture system, in which hepatocytes are sandwiched between two layers of collagen, has been shown to be capable of maintaining long-term expression of hepatocellular function (J. C. Y. Dunn et al., Biotechnol. Prog. 7, 237-245, 1991). The development of an adequate technique for the cryopreservation of hepatocytes in such a stable culture configuration would ensure a ready supply of hepatocytes for use in bioreactors or bioartificial liver support devices. This report describes the effects of exposing hepatocytes in sandwich culture to different concentrations of the cryoprotectant dimethyl sulfoxide (Me2SO) at 22 degrees C on Day 7 of culture. Cell function, morphology, and cytoskeletal organization were followed for 14 days after exposure. Hepatocellular morphology and albumin secretion remained normal when cultures were exposed for up to 120 min to predicted final Me2SO concentrations up to 1.33 M. Exposure for less than 60 min to equilibrium concentrations of up to 3.33 M Me2SO did not adversely affect cell morphology or albumin secretion rate, but at the highest concentration (3.33 M), increase of the exposure time to 60 or 120 min resulted in dramatic, irreversible cell damage and loss of function. Actin filament organization was shown to be undisturbed when the cells were exposed to 1.33 M Me2SO for 60 min, but was irreversibly disrupted by exposure to 3.33 M for 120 min. Based on these results, a simple and safe procedure is suggested for the addition of Me2SO to hepatocytes in a sandwich culture configuration and its subsequent removal, which will be valuable for studies on hepatocyte cryopreservation.

Actins↗
PubMed1992

Long-term functional recovery of hepatocytes after cryopreservation in a three-dimensional culture configuration.

Hepatocyte cryopreservation is essential to ensure a ready supply of cells for use in transplantation or as part of an extracorporeal liver assist device to provide on-demand liver support. To date, most of the work on hepatocyte cryopreservation has been performed on isolated hepatocytes, and has generally yielded cells which display low viability and greatly reduced short-term function. This report presents the development of a freezing procedure for hepatocytes cultured in a sandwich configuration. A specially designed freezing unit was used to provide controlled temperatures throughout the freeze-thaw cycle. Cooling rate, warming rate, and final freezing temperature were evaluated as to their effect on hepatocyte function as judged by albumin secretion. Under optimized conditions (cooling at 5 degrees C/min and warm at > or = 400 degrees C/min), freezing to -40 degrees C resulted in full recovery of albumin secretion within 2-3 days post-freezing, whereafter albumin secretion levels remained normal for the duration of the experiments (2 wks). Freezing to -80 degrees C lead to an approximate 70% recovery of long-term protein secretion when compared to control cultures. In addition, the overall hepatocyte morphology as judged by light microscopy, closely followed the functional results. The sandwich culture configuration, thus, enables hepatocytes to maintain a satisfactory level of long-term protein secretion after a freeze-thaw cycle under optimized conditions, and offers an attractive tool for further studies into the mechanisms of freezing injury and subsequent hepatocellular recovery. These results are a promising step in the development of satisfactory storage procedures for hepatocytes.

Animals↗
PubMed1991

Cryomicroscopic analysis of intracellular ice formation during freezing of mouse oocytes without cryoadditives.

Kinetics of intracellular ice formation (IIF) under various freezing conditions was investigated for mouse oocytes at metaphase II obtained from B6D2F1 mice. A new cryostage with improved optical performance and "isothermal" temperature field was used for nucleation experiments. The maximum thermal gradient across the window was less than 0.1 degrees C/10 mm at sample temperatures near 0 degrees C. The dependence of IIF on the initial concentration of the suspending medium was found to be pronounced. The mean IIF temperatures were found to be -9.56, -12.49, -17.63, -22.20 degrees C for freezing at 120 degrees C/min in 200, 285, 510, and 735 mosm phosphate-buffered saline, respectively. For concentrations higher than 735 mosm, the kinetics of IIF showed a break point at approximately -31 degrees C. Below -31 degrees C, all the remaining unfrozen oocytes underwent IIF almost immediately over a temperature range of less than 3 degrees C. This dramatic shift in the kinetics of IIF suggests that there were two distinct mechanisms responsible for IIF during freezing. The effect of the cooling rate on the kinetics of IIF was also investigated in isotonic PBS. At 1 degrees C/min none of the oocytes contained ice, whereas, at 5 degrees C/min all the oocytes contained ice. The mean IIF temperatures for cooling rates between 1 and 120 degrees C/min were almost constant with an average of -12.82 +/- 0.6 degrees C (SEM). In addition, constant temperature experiments were conducted in isotonic PBS. The percentages of oocytes with IIF were 0, 50, 60, and 95% for -3.8, -6.4, -7.72, and -8.85 degrees C. In undercooling experiments, IIF was not observed until approximately -20 degrees C (at which temperature the whole suspension was frozen spontaneously), suggesting the involvement of the external ice in the initiation of IIF between approximately -5 and -31 degrees C during freezing of oocytes.

Animals↗