Biomedical subjects

M Toner

Publications and source records attributed to M Toner.

At least 55 records · Page 3Linked to original sources

PubMed1999

Absence of Epstein-Barr virus encoded RNA and latent membrane protein (LMP1) in salivary gland neoplasms.

A series of 55 (42 benign and 13 malignant) salivary gland tumours were investigated by immunohistochemistry, to detect Epstein-Barr virus (EBV) latent membrane protein (LMP1) and by in situ hybridization for EBV-encoded RNA. Non-neoplastic gland from all the patients with tumours and 15 control glands were also examined. All cases, both neoplastic and non-neoplastic were negative for LMP1 and failed to show any positive signal by in situ hybridization for EBV RNA. One undifferentiated carcinoma from a European patient was included in the group. These results confirm previous reports of an ethnic association between EBV and undifferentiated carcinomas of the salivary gland. They do not support an aetiological role for EBV in other salivary gland tumours.

Adenoma↗

PubMed1999

Effects of cryoprotectants and ice-seeding temperature on intracellular freezing and survival of human oocytes.

The accurate determination of the freezing conditions that promote intracellular ice formation (IIF) is crucial for designing cryopreservation protocols for cells. In this paper, the range of temperatures at which IIF occurs in human oocytes was determined. Fresh oocytes with a germinal vesicle, failed-to-fertilize (metaphase I and metaphase II stages) and polyspermic eggs were used for this study. The occurrence of IIF was first visualized at a cooling rate of 120 degrees C/min using a programmable thermal microscope stage connected to a videomicroscope. Then, with a cooling rate of 0.2 degrees C/min, the seeding temperature of the extracellular ice was modified to decrease the incidence of IIF and increase the survival rate of frozen-thawed human oocytes. After adding different cryoprotectants, the median temperature of IIF (TMED) was decreased by approximately 23 degrees C in mouse and only by approximately 6.5 degrees C in human oocytes. Using 1.5 M propylene glycol and seeding temperatures of -8.0, -6.0 and -4.5 degrees C, the incidence of IIF was 22/28 (78%), 8/24 (33%) and 0/33 (0%) and the 24 h post-thaw survival rate was 10/31(32%), 19/34 (56%) and 52/56 (93%) respectively. The results show that IIF occurs more readily in human oocytes, and that ice seeding between -6 degrees C and -8 degrees C triggers IIF in a large number of human oocytes. Undesirable IIF can be prevented and survival rates maximized by raising the seeding temperature as close as possible to the melting point of the solution, which in our instrument was -4.5 degrees C.

Animals↗

PubMed1999

Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells.

Heterotypic cell interaction between parenchymal cells and nonparenchymal neighbors has been reported to modulate cell growth, migration, and/or differentiation. In both the developing and adult liver, cell-cell interactions are imperative for coordinated organ function. In vitro, cocultivation of hepatocytes and nonparenchymal cells has been used to preserve and modulate the hepatocyte phenotype. We summarize previous studies in this area as well as recent advances in microfabrication that have allowed for more precise control over cell-cell interactions through 'cellular patterning' or 'micropatterning'. Although the precise mechanisms by which nonparenchymal cells modulate the hepatocyte phenotype remain unelucidated, some new insights on the modes of cell signaling, the extent of cell-cell interaction, and the ratio of cell populations are noted. Proposed clinical applications of hepatocyte cocultures, typically extracorporeal bioartificial liver support systems, are reviewed in the context of these new findings. Continued advances in microfabrication and cell culture will allow further study of the role of cell communication in physiological and pathophysiological processes as well as in the development of functional tissue constructs for medical applications.

Animals↗

PubMed1999

Posttranslational modifications of cardiac and skeletal muscle proteins by reactive oxygen species after burn injury in the rat.

OBJECTIVE: To determine the involvement of oxidative damage in muscle wasting after burn injury. SUMMARY BACKGROUND DATA: Burn injury damages tissue at the site of the burn and also affects peripheral tissue. There is evidence to suggest that reactive oxygen species may be generated in increased amounts after burn, and these may contribute to wound healing and to posttranslational modifications of tissue constituents distant from the wound site. METHODS: The oxidation of muscle proteins was assessed, using the dinitrophenylhydrazine assay for carbonyl content, in muscles of rats after a full-thickness skin scald burn covering 20% of the total body surface area, over a 6-week period. In this model, rats failed to incur normal body weight or muscle weight gain. RESULTS: Soleus, extensor digitorum longus, diaphragm, and heart ventricle proteins were oxidatively damaged after injury. The extent of tissue protein oxidation, however, differed depending on the time points studied. In general, higher levels of protein carbonyl group formation, an indicator of oxidative damage, were found to occur within 1 to 5 days after injury, and the oxidized protein content of the various tissues decreased during the later stages. Both sarcoplasmic and myofibrillar carbonyl-containing proteins accumulated in diaphragm 3 days after burn injury and were rapidly removed from the tissue during a 2-hour in vitro incubation. This coincided with increased proteolytic activity in diaphragm. CONCLUSIONS: These observations suggest that the loss of proteins modified by reactive oxygen species may contribute to the burn-induced protein wasting in respiratory and other muscles by a proteolytically driven mechanism.

Animals↗

PubMed1999

Molding of deep polydimethylsiloxane microstructures for microfluidics and biological applications.

Here we demonstrate the microfabrication of deep (> 25 microns) polymeric microstructures created by replica-molding polydimethylsiloxane (PDMS) from microfabricated Si substrates. The use of PDMS structures in microfluidics and biological applications is discussed. We investigated the feasibility of two methods for the microfabrication of the Si molds: deep plasma etch of silicon-on-insulator (SOI) wafers and photolithographic patterning of a spin-coated photoplastic layer. Although the SOI wafers can be patterned at higher resolution, we found that the inexpensive photoplastic yields similar replication fidelity. The latter is mostly limited by the mechanical stability of the replicated PDMS structures. As an example, we demonstrate the selective delivery of different cell suspensions to specific locations of a tissue culture substrate resulting in micropatterns of attached cells.

Biocompatible Materials↗

PubMed1999

Numerical model of fluid flow and oxygen transport in a radial-flow microchannel containing hepatocytes.

The incorporation of monolayers of cultured hepatocytes into an extracorporeal perfusion system has become a promising approach for the development of a temporary bioartificial liver (BAL) support system. In this paper we present a numerical investigation of the oxygen tension, shear stress, and pressure drop in a bioreactor for a BAL composed of plasma-perfused chambers containing monolayers of porcine hepatocytes. The chambers consist of microfabricated parallel disks with center-to-edge radial flow. The oxygen uptake rate (OUR), measured in vitro for porcine hepatocytes, was curve-fitted using Michaelis-Menten kinetics for simulation of the oxygen concentration profile. The effect of different parameters that may influence the oxygen transport inside the chambers, such as the plasma flow rate, the chamber height, the initial oxygen tension in the perfused plasma, the OUR, and K(m) was investigated. We found that both the plasma flow rate and the initial oxygen tension may have an important effect upon oxygen transport. Increasing the flow rate and/or the inlet oxygen tension resulted in improved oxygen transport to cells in the radial-flow microchannels, and allowed significantly greater diameter reactor without oxygen limitation to the hepatocytes. In the range investigated in this paper (10 microns < H < 100 microns), and for a constant plasma flow rate, the chamber height, H, had a negligible effect on the oxygen transport to hepatocytes. On the contrary, it strongly affected the mechanical stress on the cells that is also crucial for the successful design of the BAL reactors. A twofold decrease in chamber height from 50 to 25 microns produced approximately a fivefold increase in maximal shear stress at the inlet of the reactor from 2 to 10 dyn/cm2. Further decrease in chamber height resulted in shear stress values that are physiologically unrealistic. Therefore, the channel height needs to be carefully chosen in a BAL design to avoid deleterious hydrodynamic effects on hepatocytes.

Animals↗

PubMed1998

Prevention of hemolysis in rapidly frozen erythrocytes by using a laser pulse.

This paper reports on the successful recovery of rapidly frozen unprotected erythrocytes by vitrification of the intracellular solution with a laser pulse prior to thawing. Erythrocytes that were frozen at 10,000 degrees C/min exhibited 100% hemolysis when thawed unless they were first irradiated by a 7 ns. laser pulse that selectively targeted the intracellular ice so that it was melted and resolidified into a glass phase. Up to 80% of the cells treated in this way remained intact after thawing. Wright's staining confirmed a healthy cell morphology and the retention of hemoglobin in the laser treated cells. While it is well known that small amounts of intracellular ice can be tolerated by cells, the findings of this study are the first to indicate that intracellular ice may be innocuous even when formed in substantial quantities provided that crystal growth and coalescence can somehow be avoided during warming.

Blood Preservation↗

PubMed1998

Poloxamer 188 enhances functional recovery of lethally heat-shocked fibroblasts.

Damage to the cell membrane has been implicated as the primary event in the pathogenesis of heat shock, generally resulting in loss of cellular homeostasis and cell death. Thus a promising mode of therapy would involve the restoration of cell membrane integrity. Surfactant molecules, specifically triblock polymers such as Poloxamer 188 (P-188), possess the ability to self-aggregate into membrane-like structures in aqueous solutions and have been shown to restore membrane integrity. The objective of this study was to develop functional and morphological assays to determine whether treatment with P-188 after heat shock enhances the recovery of thermally damaged cells. Human foreskin fibroblasts were placed in sterile vials and heated by immersion in a calibrated water bath for various lengths of time at predefined temperatures. Cell recovery after heat shock was assessed using a functional assay based on the ability of the cells to contract fibroblast populated collagen lattices (FPCLs). Subsequent to heating, collagen lattices were prepared with control (no heat, no P-188) and heat shocked cells (with and without P-188). Our results indicate that treatment with low concentrations of P-188 after heat shock was effective in ameliorating both the morphological integrity and the contractile function of thermally damaged cells. Further, we observed that P-188 was most effective in improving the contractile ability of cells heat shocked at 45 degrees C; however, it had no influence on the contractility of cells exposed to higher temperatures. Our results suggest that there exists a threshold of thermal stress (45 degrees C for 20-60 min) beyond which treatment with low concentrations of P-188 (0.5 mg/ml) is ineffective in minimizing cell damage. Moreover, the results of our morphological assays indicate that cells treated with P-188 after heat shock maintain their cytoskeletal organization, whereas untreated cells exhibit filamentous actin depolymerization.

Actin Cytoskeleton↗

PubMed1998

Interaction between heat shock and interleukin 6 stimulation in the acute-phase response of human hepatoma (HepG2) cells.

Two characteristic elements of the acute-phase response are an altered pattern of circulating hepatic proteins and fever. Whereas a fever-induced heat shock response could affect expression of acute-phase proteins in the liver, the effects of a modest temperature increase on protein secretion in interleukin-6 (IL-6)-stimulated HepG2 cells were investigated. The response of HepG2 cells to IL-6 stimulation was significantly affected by heat treatment at 40 degreesC. Albumin secretion rates, which were reduced by a factor of 2 in response to either heat shock or IL-6 stimulation alone, were down-regulated by a factor of 4 when IL-6 was administered simultaneously with a continuous 40 degrees C heat shock. IL-6-induced fibrinogen up-regulation was significantly reduced by heat treatment (P < .01), and secretion rates were indistinguishable from control levels after 2 days (P > .10). Unexpectedly, heat shock at 40 degrees C induced a fivefold up-regulation of haptoglobin production in the absence of IL-6. Simultaneous heat shock and IL-6 stimulation caused a synergistic enhancement of haptoglobin expression, with secretion rates increasing up to 30-fold compared with unstimulated control cells. For all three proteins, the interaction between temperature and IL-6 concentration was statistically significant (P < .001). Heat treatment resulted in significant alterations of both the kinetics and sensitivity of IL-6-induced protein synthesis, suggesting a major modification of the mechanism of acute-phase protein regulation at 40 degreesC. In summary, the data show that heat shock can significantly modulate the pattern of acute-phase protein expression and that fever may be an important regulatory factor in the acute-phase response.

Acute-Phase Proteins↗

PubMed1998

Cytoskeleton and polyploidy after maturation and fertilization of cryopreserved germinal vesicle-stage mouse oocytes.

PURPOSE: Our purpose was to assess the effect of cryopreservation on cytoskeleton of germinal vesicle (GV) mouse oocytes and determine whether irreversible spindle damage and related digyny associated with cryopreservation of metaphase II (MII) oocytes can be avoided. METHODS: The GV oocytes were cryopreserved using a slow-cooling (0.5 degree C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethylsulfoxide supplemented with 0.2 M sucrose and analyzed before and during fertilization by multiple-label fluorescence and differential interference contrast microscopy techniques. RESULTS: When examined after in vitro maturation, the vast majority (> 95%) of cryopreserved and control oocytes displayed normal microfilament and microtubule organization. With respect to barrel-shaped spindle and normal chromosome alignment, no significant differences were observed between cryopreservation (78 and 86%, respectively) and control (85 and 95%, respectively) groups. In fertilization experiments, spindle rotation, formation of the second polar body, and pronuclear migration were displayed by similar percentages of cryopreserved (96, 94, and 37%, respectively) and control (98, 97, and 45%, respectively) oocytes, indicating normal functionality of the cytoskeleton during this period. However, pronuclear formation was significantly inhibited by cryopreservation (81%) compared with controls (100%). Regarding digyny and polyspermy, no significant increase was observed after cryopreservation (3 and 10%, respectively) compared with controls (3 and 6%, respectively). CONCLUSIONS: Cryopreservation of mouse oocytes at the GV stage is particularly advantageous to circumvent the spindle damage and increased digyny noted after cryopreservation of MII oocytes.

Actin Cytoskeleton↗

PubMed1998

CT and MRI appearances of a thoracic chordoma.

A case of a chordoma in the thoracic spine is presented. This is a very rare tumour in this location and should be considered in the differential diagnosis of any posterior mediastinal mass. The appearances on CT and MRI were similar to chordomas described in other locations. On T2-weighted images septae of low signal intensity radiated throughout the large high-signal mass. This feature may be of use in differentiating chordomas from other posterior mediastinal masses.

Adult↗

PubMed1998

A theoretical formalism for aggregation of peroxidized lipids and plasma membrane stability during photolysis.

The objective of this investigation was to examine, from a theoretical perspective, the mechanism underlying the lysis of plasma membranes by photoinduced, chemically mediated damage such as is found in photolysis. Toward this end, a model is presented which relates the membrane lifetime to the thermodynamic parameters of the membrane components based upon the kinetic theory of aggregate formation. The formalism includes a standard birth/death process for the formation of damaged membrane components (i.e., peroxidized lipids) as well as a terminating condensation process for the formation of aggregates of peroxidized plasma membrane lipids. Our theory predicts that 1) the membrane lifetime is inversely correlated with predicted rate of membrane damage; 2) an upper limit on the duration of membrane damage exists, above which the mean and variance of the membrane lifetime is independent of further membrane damage; and 3) both the mean and variance of the time of membrane lifetime distribution are correlated with the number of sites that may be damaged to form a single membrane defect. The model provides a framework to optimize the lysis of cell membranes by photodynamic therapy.

Biophysical Phenomena↗

PubMed1998

Alterations of the cytoskeleton and polyploidy induced by cryopreservation of metaphase II mouse oocytes.

OBJECTIVE: To determine cryopreservation-induced alterations in the cytoskeleton of metaphase II mouse oocytes and the implications of these alterations in functionality of the cytoskeleton and polyploidy after fertilization. DESIGN: Comparative study. SETTING: Clinical and academic research environment at a medical school teaching hospital. INTERVENTION(S): Oocytes were frozen using a slow-cooling (0.5 degrees C/min) and slow-thawing (8 degrees C/min) protocol in 1.5 M dimethyl sulfoxide and 0.2 M sucrose and were analyzed before and after fertilization. MAIN OUTCOME MEASURE(S): Cytoskeletal alterations, fertilization, and polyploidy rates. RESULT(S): When analyzed immediately after thawing, the oocytes displayed dramatic cytoskeletal alterations. Only slight recovery was observed upon removal of the cryoprotectants. However, incubation after thawing of 1 hour at 37 degrees C completely reestablished a normal microfilament and microtubule pattern while partially restoring normal spindle morphology and chromosome alignment. Accordingly, insemination immediately after removal of cryoprotectants resulted in a significantly decreased fertilization rate and aberrant dynamics of cytoskeleton-dependent events, whereas oocytes inseminated after the post-thaw incubation displayed fertilization rates and cytoskeletal dynamics comparable to those in controls. Cryopreservation did not increase polyspermy but significantly increased digyny when the oocytes were inseminated after the post-thaw incubation. All digynic eggs displayed an abnormal spindle remnant in comparison with diploid or polyspermic eggs. CONCLUSION(S): A brief period of incubation after thawing allows recovery and positively affects fertilization and cytoskeletal dynamics. Cryopreservation does not impair the functionality of microfilaments and cytoplasmic microtubules during postfertilization events. Our findings suggest that the increased rate of digyny in cryopreserved oocytes may be related to the spindle disorganization, leading to failure in segregation of the chromosomes, rather than to direct malfunction of the microfilaments in polar body formation.

Animals↗

PubMed1998

Intracellular calcium dynamics during photolysis.

The objective of this investigation was to gain a deeper understanding of the intracellular events that precede photolysis of cells. A model system, consisting of malignant melanoma cells pretreated with the calcium sensitive fluorescent dye, Fluo-3, was used to examine the intracellular calcium dynamics in single-cell photolysis experiments. Exposure of the cells to 632 nm laser light in the presence of photosensitizer, tin chlorin e6, resulted in a rise in intracellular calcium. The increase in intracellular calcium was blocked using a variety of calcium channel blocking agents, including verapamil, nifedipine, and nickel. Treatment with the channel blockers was also effective in either decreasing or eliminating cell death despite the presence of lethal doses of photosensitizer and irradiation. These results show that intracellular calcium rises prior to plasma membrane lysis, and that this early rise in intracellular calcium is necessary for membrane rupture.

Aniline Compounds↗

PubMed1998

Probing heterotypic cell interactions: hepatocyte function in microfabricated co-cultures.

Replacement of liver function using extracorporeal bioartificial systems has been attempted with limited success. The instability of the hepatocyte phenotype in vitro has restricted the useful lifetime of these devices. Co-cultivation of hepatocytes with mesenchymal cells is one method that has been widely utilized to stabilize the liver-specific function of isolated cells; however, co-culture has yet to be successfully incorporated in a bioreactor setting. In this study, we probed heterotypic cell interactions in co-cultures of hepatocytes and 3T3 in order to better understand the cellular microenvironment necessary to induce and stabilize liver-specific functions. Using microfabrication and conventional techniques to control the heterotypic interface, the effects of varying degrees of heterotypic interaction on tissue function (albumin and urea synthesis) were examined. Our data indicated maximal induction of liver-specific functions in cultures with maximal initial heterotypic interaction, and that induction of hepatic functions in hepatocytes was increased in the vicinity of fibroblasts as compared to hepatocytes far from the heterotypic interface. Furthermore, our data suggested that heterotypic cell contact is necessary for induction of these functions. These studies will aid in the formation of design criteria for a co-culture based bioartificial liver, as well as provide a useful tool to study the role of heterotypic and homotypic interactions in liver physiology and pathophysiology.

3T3 Cells↗

PubMed1997

Controlling cell interactions by micropatterning in co-cultures: hepatocytes and 3T3 fibroblasts.

The repair or replacement of damaged tissues using in vitro strategies has focused on manipulation of the cell environment by modulation of cell-extracellular matrix interactions, cell-cell interactions, or soluble stimuli. Many of these environmental influences are easily controlled using macroscopic techniques; however, in co-culture systems with two or more cell types, cell-cell interactions have been difficult to manipulate precisely using similar methods. Although microfabrication has been widely utilized for the spatial control of cells in culture, these methods have never been adapted to the simultaneous co-cultivation of more than one cell type. We have developed a versatile technique for micropatterning of two different cell types based on existing strategies for surface modification with aminosilanes linked to biomolecules and the manipulation of serum content of cell culture media. This co-culture technique allowed manipulation of the initial cellular microenvironment without variation of cell number. Specifically, we were able to control the level of homotypic interaction in cultures of a single cell type and the degree of heterotypic contact in co-cultures over a wide range. This methodology has potential applications in tissue engineering, implant biology, and developmental biology, both in the arena of basic science and optimization of function for technological applications.

3T3 Cells↗

PubMed1997

Identifying stage B colorectal cancer patients at high risk of tumor recurrence and death.

PURPOSE: This study was designed to determine clinical and pathologic variables associated with poor outcome following resection of Stage B colorectal cancer. METHODS: This was a retrospective study of 117 patients with Stage B cancer who underwent curative surgery and survived the postoperative period. Fourteen clinical and pathologic features were studied. Clinical data were extracted from a prospective colorectal cancer database, and histologic slides were retreived and examined by a pathologist blinded as to clinical details and outcome. RESULTS: After a median follow-up period of 8.2 years, bowel obstruction was significantly related to a poor prognosis (log-rank test; P = 0.03). Extensive necrosis (P = 0.01) and perineural invasion (P = 0.03) were also associated with decreased survival. Vascular invasion was associated with poor long-term outcome in the subgroup of patients with rectal (P = 0.07) but not colonic (P = 0.57) cancer. Multivariate regression analysis identified both tumor necrosis (P = 0.01) and perineural invasion (P = 0.03) as independently related to outcome. CONCLUSION: Further study of prognostic indicators might result in an algorithm to distinguish Stage B cases at high risk of tumor recurrence and death. Such patients could be included in future trials of adjuvant therapies.

Adult↗

PubMed1997

Cavernous hemangioma in the submandibular gland masquerading as sialadenitis: case report.

This report describes a case of cavernous hemangioma in the submandibular gland of a 37-year-old Irish woman. The lesion clinically and radiologically (as shown both by plain radiographs and computed tomography imaging) resembled salivary calculous disease. Numerous phleboliths were present in the hemangioma. The rarity of a cavernous hemangioma in such a location and its simulation of salivary calculous disease stimulated us to report the case.

Adult↗